Synthesis of the trisaccharide repeating unit of capsular polysaccharide from Klebsiella pneumoniae

Article abstract of DOI:10.1016/j.tetlet.2018.12.031

The incidences of primary, pyrogenic liver abscess complicated by meningitis and septic endophthalmitis caused by Klebsiella pneumoniae has increased globally. Earlier studies have shown that the capsular polysaccharide (CPS) of Klebsiella pneumoniae plays an important role in the resistance to phagocytosis and related pathogenicity. The first chemical synthesis of the trisaccharide repeating unit of this CPS has been achieved via stereoselective glycosylation with orthogonal and appropriate protecting groups. A new method for the pyruvylation of trans diol was developed.

Full text of DOI:10.1016/j.tetlet.2018.12.031

Accepted Manuscript
Synthesis of the Trisaccharide Repeating Unit of Capsular Polysaccharide from
Klebsiella Pneumoniae
Woen Susanto, Kah-Hoe Kong, Kuo-Feng Hua, Shih-Hsiung Wu, Yulin Lam
PII:
S0040-4039(18)31473-4
https://doi.org/10.1016/j.tetlet.2018.12.031
TETL 50494
DOI:
Reference:
Tetrahedron Letters
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13 November 2018
8 December 2018
14 December 2018
Please cite this article as: Susanto, W., Kong, K-H., Hua, K-F., Wu, S-H., Lam, Y., Synthesis of the Trisaccharide
Repeating Unit of Capsular Polysaccharide from Klebsiella Pneumoniae, Tetrahedron Letters (2018), doi: https://
doi.org/10.1016/j.tetlet.2018.12.031
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Synthesis of the Trisaccharide Repeating
Unit of Capsular Polysaccharide from
Klebsiella Pneumoniae
Woen Susanto, Kah-Hoe Kong, Kuo-Feng Hua,
Shih-Hsiung Wu,* and Yulin Lam*
The first reported synthesis of the
trisaccharide repeating unit of capsular
polysaccharide from Klebsiella
Pneumoniae

1
Tetrahedron Letters
Synthesis of the Trisaccharide Repeating Unit of Capsular Polysaccharide from
Klebsiella Pneumoniae
Woen Susanto,a Kah-Hoe Kong,a Kuo-Feng Hua,b Shih-Hsiung Wu,* c and Yulin Lam* a
a Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
b Department of Biotechnology and Animal Science, National Ilan University, No 1 Sec 1 Shennong Road, Yilan County 260, Taiwan
c Institute of Biological Chemistry, Academia Sinica, 128 Academia Road Sec 2, Nankang, Taipei 115, Taiwan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The incidences of primary, pyrogenic liver abscess complicated by meningitis and septic
endophthalmitis caused by Klebsiella Pneumoniae has increased globally. Earlier studies have
shown that the capsular polysaccharide (CPS) of Klebsiella Pneumoniae plays an important role
in the resistance to phagocytosis and related pathogenicity. The first chemical synthesis of the
trisaccharide repeating unit of this CPS has been achieved via stereoselective glycosylation with
orthogonal and appropriate protecting groups. A new method for the pyruvylation of trans diol
was developed.
Available online
Keywords:
Trisaccharide
CPS
2009 Elsevier Ltd. All rights reserved.
Pyruvylation
Klebsiella Pneumoniae
Chemical synthesis
1. Introduction
and pyruvyl groups which in turn resulted in the loss of
immunomodulatory activities.
Klebsiella pneumonia (K. pneumonia) is a gram-negative
pathogen of the Enterbacteriaceae family which causes
opportunistic infections, such as pneumonia, sepsis and
inflammation of the urinary tract.1 In the past two decades, the
incidence of K. pneumonia-caused primary pyrogenic liver
abscess (PLA), complicated by meningitis or septic
endophthalmitis, has increased globally.2 Despite advances in
medical care, PLA is still a critical disease with high morbidity
and mortality rates.3 Hence there is an impetus to develop
effective therapies for protection against such bacterial
infections.
We envisaged that chemical synthesis would be a viable
alternative to obtain well-defined short chain CPS and its
structural analogs for structure-activity relationship studies and to
determine the minimal epitope requirements to elicit protective
immune responses.7 To address these questions, we report herein
the first chemical synthesis of the K. pneumonia CPS
trisaccharide repeating unit 1. The preparation of 1 would itself
be interesting too because of the unusual (S)-configurated
pyruvic acid acetal group in the 2,3-O-positions of D-glucuronic
acid. Although various methods have been reported for the
pyruvylation of cis-vicinal diols,8 but methods to prepare pyruvic
acid linked to vicinal trans-positions are less common.9 Hence we
herein also describe a new method for the pyruvylation of trans
diol.
Earlier studies have revealed that the capsular polysaccharide
(CPS) of K. pneumonia played an important role in the virulence
and pathogenesis of the bacteria4 and animal studies have shown
that rats which are actively immunized with the purified CPS
appeared to be protected against the lethal pneumonia caused by
K. pneumonia.4b,5 Recently we6 have shown that the CPS of PLA
K. pneumonia (MW ≥ 500 000) consists of repeating units of the
trisaccharide
(3)--D-Glc-(14)-[2,3-(S)-pyruvate]--D-
GlcA-(14)--L-Fuc-(1) 1 which has an unusual feature of
pyruvylation on glucuronic acid and acetylation of either the C-2
or C-3 -OH, but not both hydroxyl groups of fucose (Figure 1).
We have also demonstrated that CPS induces secretion of tumor
necrosis factor- (TNF-) and interleukin-6 from wild-type
macrophages through Toll-like receptor 4 (TLR4). To determine
if short chain CPS would retain the immunomodulatory
properties, we attempted to digest the polymeric CPS under mild
acidic conditions. However this effected the removal of the acetyl
Fig. 1 Capsular polysaccharide of K. pneumonia
2. Result and Discussion
A retrosynthetic analysis of 1 is depicted in Scheme 1. The
key synthetic consideration is the control of anomeric
 Corresponding author. e-mail: shwu@gate.sinica.edu.tw (S H Wu); email: chmlamyl@nus.edu.sg (Y Lam)

2
stereochemistry during the respective glycosylations with or
was first deprotected and acetylated to yield compound 10. The
anomeric thiophenol protecting group was then cleaved, and the
trichloroacetimidate auxiliary attached to furnish precursor 12.
Similarly precursor 3 was transformed to the protected 13 which
could be readily synthesized from D-glucose 6 as previously
reported11 (Scheme 2). We decided to introduce an acetyl ester at
the C-2 position of precursor 13 to facilitate formation of the -
conformer via neighbouring group participation12 upon
glycosylation with the glucuronic acid fragment; this acetyl
group can subsequently be cleaved to release the free C-2
hydroxyl group required in the target trisaccharide 1.
without the participation of the protecting group at C-2.
Furthermore, it was envisaged that all the hydroxyl groups in the
saccharides would be protected with benzyl groups prior to the
final global deprotection step by catalytic hydrogenation which
would provide access to 1. On the basis of these considerations,
three building blocks 2 – 4 were designed to incorporate the O-
acetyl (4) and O-pyruvate groups (3) on the fucose and
glucoronic acid, respectively. Both 2 and 3 were installed with
glycosyl auxillary to facilitate in glycosylation. Further
transformation gave the commercially available building blocks 5
– 7.
Scheme 1 Retrosynthesis of trisaccharide 1.
Scheme 3 Synthesis of glucuronic acid fragment 24
The synthesis of the glucuronic acid fragment 7 is depicted in
Scheme 3. D-glucose was first protected as a benzyl ether at the
anomeric position, followed by C-4 and C-6 protection as the
benzylidene acetal, to yield compound 15. This was further
protected at C-2 and C-3 with 2-(bromomethyl)naphthalene to
provide compound 16. Next, acidic hydrolysis of the acetal group
liberated the C-6 primary alcohol for selective TEMPO/BAIB
oxidation,13 which was subsequently methylated to furnish
compound 18. Subsequent acetylation of the C-4 hydroxyl group
and removal of the C-2 and C-3 methylnapthyl ethers furnished
compound 20, which was the key trans-diol intermediate for the
investigation of the pyruvylation reaction. Initial attempts to form
the acetal directly from methyl pyruvate failed to provide the
desired pyruvate compound.8a Various Lewis acids (TMSOTf,
Scheme 2 Synthesis of fucose fragment 12 and glucose
fragment 13
The forward synthesis of the fucose fragment 5 was modified
to a protected fucose 8 to ensure selective glycosylation on the C-
1 position as shown in Scheme 2. Intermediate 8 was synthesized
from D-fucose according to the procedure reported by Szabó et
al.10 Since the C-2 or C-3 acetyl group is important for the
compound’s biological effects, the C-3 methylnaphthalene group

3
SO2Cl2-TfOH, CF3SO3CH3, dimethyl(methylthio) sulfonium
trifluoromethanesulfonate (DMTST), nitroso tetrafluoroborate
(NOBF4), and BF3.OEt2) were applied to the reaction but none of
them yielded the desired compound. With little success on the
direct pyruvylation of the trans-diol, we decided to use
acetoxyacetone as a precursor. To effect the transformation, we
activated the hydroxyl groups by protecting them with a silyl
group (Scheme 3, compound 21). With these changes, formation
of the acetal proceeded readily to give compound 22 in very good
yield. Subsequently, the acetyl protecting groups were removed
to form 23. A selective oxidation of the primary alcohol into
carboxylic acid followed by a methyl esterification of the acid
furnish the precursor 24.
addition, to confirm the absolute configuration of the CH3 on
the pyruvate ring, monomer 24 was initially subjected to 2D
experiments but no conclusive results were achieved. Increasing
the steric bulk on the ester by changing the methyl ester to benzyl
ester (Scheme 5) improved the degree of interactions, resulting in
successful 2D experiments. The proton at the C-3 position could
be derived with the use of COSY followed by NOESY, allowing
an interaction between the C-3 proton and the –CH3 of the
pyruvate group to be observed (See Supporting Information).
With the individual carbohydrate fragments synthesized, we
proceeded to glycosylate them. Compounds 13 and 24 were
glycosylated to give disaccharide 25 in good selectivity and yield
(Scheme 4). With an acetyl directing group at C-2 of compound
13, glycosylation gave only the -anomer of the disaccharide.
Thereafter, the ester groups on 25 were hydrolyzed. The
carboxylic acid groups were subsequently protected with a
benzyl ester 26 so as to enable global deprotection via catalytic
hydrogenation in the final step. Next, dibutyltin oxide in the
presence of benzyl bromide was used to benzylate either the C-2
or C-3 hydroxy group, but not both. This reaction provided
compound 27 and the C-3 benzylated product in 58% and 35%
yield respectively. Compound 27 was subsequently glycosylated
with fucose 12 to afford the protected trisaccharide 28 in good
stereoselectivity. With the protected trisaccharide 28 synthesized,
global deprotection of the remaining benzyl ether groups using
Pd/C in the presence of hydrogen gas gave trisaccharide 1.
Scheme 5 Synthesis of glucuronic acid fragment 29
Conclusions
In summary, we report the synthesis of the trisaccharide unit
of CPS from the liver abscess strain K. pneumoniae. The
synthetic transformations involved three building blocks 12, 13
and 24 which were eventually glycosylated into trisaccharide 1.
The overall efficiency of the assembly process benefitted from a
set of appropriately selected protecting groups. Complete
stereoselectivity for each glycosylation was achieved via the
orthogonality and participating effects of the blocking groups. In
the course of this synthesis, we have also demonstrated a new
methodology to effect pyruvylation of trans-diol.
Acknowledgments
The authors thank the Ministry of Education (Singapore) for
the financial support provided (MoE Tier 2 Grant: R-143-000-
589-112).
References and notes
1
2
(a) R. Podschun, U. Ullmann, Clin. Microbiol. Rev. 11 (1998) 589-603.
(b) R. Ramphal, P. G. Ambrose, Clin. Infect. Dis. 42 (2006) S164-S172.
(a) Y. Kohayagawa, K. Nakao, M. Ushita, N. Niino, M. Koshizaki, Y.
Yamamori, Y. Tokuyasu, H. Fukushima, J. Infect. Chemother. 15 (2009)
248-251.
(b) L. K. Siu, K. M. Yeh, J. C. Lin, C. P. Fung, F. Y. Chang, Lancet
Infect. Dis. 12 (2012) 881-887.
3
4
W. Chen, C. H. Chen, K. L. Chiu, H. C. Lai, K. F. Liao, Y. J. Ho, W. H.
Hsu, Crit. Care Med. 36 (2008) 1184-1188.
(a) S. Zamze, L. Martinez-Pomares, H. Jones, P. R. Taylor, R. J. Stillion,
S. Gordon, S. Y. Wong, J. Biol. Chem. 277 (2002) 41613-41623.
(b) C. T. Fang, Y. P. Chuang, C. T. Shun, S. C. Chang, J. T. Wang, J.
Exp. Med. 199 (2004) 697-705.
5
6
S. J. Jr. Cryz, E. Fürer, R. Germanier, Infect. Immun. 54 (1986) 403-407.
F. L. Yang, Y. L. Yang, P. C. Liao, J. C. Chou, K. C. Tsai, A. S Yang, F.
Sheu, T. L. Lin, P. F. Hsieh, J. T. Wang, K. F. Hua, S. H. Wu, J. Biol.
Chem. 24 (2011) 21041-21051.
7
(a) R. D. Astronomo, D. R. Burton, Nat. Rev. Drug Discov. 9 (2010)
308-324.
8
(a) L. G. Bennett, C. T. Bishop, Immunochem. 14 (1977) 693-696.
(b) K. Hiruma, J. Tamura, H. Hashinoto, Carbohyd. Res. 282 (1996) 299-
306.
Scheme 4 Synthesis of trisaccharide 1 from the individual
carbohydrate fragments.
(c) I. Bajza, J. Kerékgyárto, J. Hajkó, L. Szilágyi, A. Lipták, Carbohyd.
Res. 253 (1994) 111-120.
(d) H. Lõnn, Carbohyd. Res. 139 (1985) 105-113.
(e) P. Fugedi, P. J. Garegg, Carbohyd. Res. 149 (1986) C9-C12.
(f) V. Pozsgayand, J. Jennings, J. Org. Chem. 52 (1987) 4635-4637.
(g) T. Ziegler, E. Ekhardt, G. Herold, Tetrahedron Lett. 33 (1992) 4413-
4416.
The stereochemistry of 1 could not be determined accurately
as the compound exist as an anomeric mixture when both the α
and β form of the C1 hydroxy are present. As a result, 2D
experiments such as COSY, HSQC and HMBC (supporting
information) could only be performed on the anomerically-pure
intermediate 28 where the C1 hydroxy remained benzylated. In
9
C. L. Pereira, A. Geissner, C. Anish, P. H. Seeberger, Angew. Chem. Int.
Ed. Engl., 2015, 54, 10016-10019.
 Corresponding author. e-mail: shwu@gate.sinica.edu.tw (S H Wu); email: chmlamyl@nus.edu.sg (Y Lam)

4
10 Z. B. Szabó, A. Borbás, I. Bajza, A. Lipták, Tetrahedron: Asymmetry,
2005, 16, 83-95
11 (a) B. Yang, K. Yoshida, Z. Yin, H. Dai, H. Kavunja, M. H. El-
Dakdouki, S. Sungsuwan, S. B. Dulaney, X. Huang, Angew. Chem. Int.
Ed. Engl., 2012, 51, 10185-10189.
(b) P. Peng, D.-C. Xiong, X.-S. Ye, Carbohydr. Res., 2014, 384, 1-8.
12 G. Wulff, G. Rohle, Angew. Chem. Int. Ed. Engl., 1974, 13, 157-170.
13 J. D. C. Codée, B. Stubba, M. Schiattarella, H. S. Overkleeft, C. A. A.
van Boeckel, J. H. van Boom, G. A. van der Marel. J. Am. Chem. Soc.,
2005, 127, 3767-3773.
Supplementary Material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2018.xx.xxx

Highlights:



synthesis of the trisaccharide repeating unit of CPS of K. Pneumoniae
a new method for the pyruvylation of trans diol
stereoselective glycosylation with orthogonal protecting groups