Synthesis of galabiose-chitosan conjugate as potent inhibitor of streptococcus suis adhesion

Article abstract of DOI:10.1021/bm100289v

The aim of this work is to construct a safe and effective drug candidate against Streptococcus suis infection. A panel of chitosan-based polymer conjugates with branched galabiose (Galα1-4Gal) side chains was synthesized as inhibitors of S. suis adhesion.

Full text of DOI:10.1021/bm100289v

Biomacromolecules 2010, 11, 1701–1704
1701
Synthesis of Galabiose-chitosan Conjugate as Potent Inhibitor
of Streptococcus suis Adhesion
†,‡
§
§
†
,†
Yaozu Xu, Hongjie Fan, Chengping Lu, George F. Gao, and Xuebing Li*
Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy
of Sciences, Beijing 100190, China, Graduate University of Chinese Academy of Sciences,
Beijing 100049, China, and Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of
Agriculture, Nanjing Agricultural University, Nanjing 210095, China
Received March 17, 2010; Revised Manuscript Received June 2, 2010
The aim of this work is to construct a safe and effective drug candidate against Streptococcus suis infection. A
panel of chitosan-based polymer conjugates with branched galabiose (GalR1-4Gal) side chains was synthesized
as inhibitors of S. suis adhesion. The synthesis was achieved by using an aldehyde-functionalized galabiose
1
derivative to graft it onto chitosan amino groups. Structural compositions of the conjugates were verified by H
NMR spectroscopy and CHN elemental analyses. Potent inhibitory activities of the conjugates against S. suis
adhesion to human erythrocytes were determined at low nanomolar concentration by HAI assay. An SPR study
revealed a high affinity binding (K
d
4
) 39.6 nM) of the conjugate with BSI-B lectin. By using biocompatible
chitosan as the scaffold for presenting S. suis-specific galabiose units, as well as the concise route tailored for the
conjugate syntheses, the present study provides a practical way for explorations of new anti-S. suis therapies.
Introduction
To meet the goal of developing safe and practical drug
candidates against S. suis infection, here we report the facile
synthesis of a new anti-S. suis agent, galabiose-chitosan (GC)
conjugate 1 (Scheme 1), which has a chitosan backbone with
branched galabiose side chains, and also demonstrate its ability
to inhibit S. suis adhesion at low nanomolar concentration. It is
advantageous to use chitosan as the scaffold for displaying
multivalent galabiose residues because (i) chitosan is an
abundant natural polysaccharide with amino groups that allow
easy chemical modifications; (ii) chitosan has shown a broad-
Streptococcus suis is a serious zoonotic pathogen causing
meningitis, septicemia, and pneumonia in both pigs and human.
The repeated worldwide outbreaks of S. suis infection with high
mortality, up to 17.8% in human cases, pinpoint the urgent need
for safe and effective methods for microbial prevention and
treatment. Antibiotics such as penicillin G and gentamicin are
powerful tools in the fight against this contagious pathogen.
However, the effectiveness of traditional antibiotics is under
1
2
8
threat from the alarming increase in bacterial resistance. A
recent alternative strategy termed antiadhesion therapy is to
spectrum antibacterial activity toward various species; and (iii)
3
chitosan is biodegradable, biocompatible, and has been applied
9
use agents that interfere with microbial attachment to host
tissues. Adhesion is the first key step in the infection process
that in many cases is mediated by the specific binding of
bacterial lectinlike proteins (adhesins) to complementary glycan
receptors present on the host cell’s surface. Creation of
to medical areas such as wound healing. The unique combina-
tion of S. suis-specific galabiose with diverse chitosan bioac-
tivities prompts us to start the synthetic and bioevaluation
research program.
4
multivalent glyco-mimics with high affinity for the bacterial
Experimental Section
surface proteins is by far one of the most promising approaches
based on antiadhesion strategy. Since the first identification of
carbohydrate receptor, galabiose (GalR1-4Gal), of S. suis in
993, several studies have been conducted to develop galabiose
derivatives as S. suis blockers. In these reports, enhanced
Materials. Five types of chitosans with the same degree of
deacetylation (DDA, 95% GlcN, 5% GlcNAc) but different average
molecular weight (M , 5500, 1000, 300, 100, and 50 kDa, respectively)
w
5
1
were purchased from Zhejiang Golden-shell Biochemical Co., Ltd. The
molecular weight distribution of chitosans was generally moderate, with
a polydispersity index (PDI) at a range of 1.4-2.2. A galactose-specific
6
inhibitory effect of the multivalent galabiosides as low as
nanomolar concentrations was found by clustering galabiose
7
10
virulent S. suis type 2 strain (HA9801, a vaccine strain in China),
isolated from diseased pigs in Jiangsu province of China, was used in
this study. The strain was grown in Todd-Hewitt broth (THB) or agar
medium and supplemented with 2% (V/V) newborn bovine serum at 37
residue on the synthetic scaffolds such as aminoethoxyl benzoic
acid and poly(amidoamine) dendrimer, although the preparations
of such dendrimer blockers were complicated, involving mul-
tistep modifications of the galabiose unit as well as generations
of the scaffolds, and therefore, could hardly meet the require-
ment of drug safety and practical applications to produce an
abundant supply.
°
C. Human erythrocytes (blood group B) were obtained from General
Hospital of China People’s Liberation Army. BSI-B lectin (Bandeiraea
4
simplicifolia) was purchased from Sigma Co., Ltd. Compounds 3, 5,
and 6 were synthesized as described in Supporting Information.
General Procedure for the Synthesis of GC Conjugate
(
1). Ozonolysis of compound 6 in methanol (0.1 mL/mg of 6) at -78
C quantitatively yielded aldehyde 7. A reaction mixture for conjugate
*
To whom correspondence should be addressed. Tel.: +86-10-62526982.
°
Fax: +86-10-62526982. E-mail: lixb@im.ac.cn.
†
syntheses was composed of chitosan (2.0 mg/mL), aldehyde 7 (0.1-2.0
equiv of chitosan GlcN), and sodium cyanoborohydride (2 equiv of 7)
in an aqueous 5 wt % acetic acid solution. The mixture was stirred for
Chinese Academy of Sciences.
Graduate University of Chinese Academy of Sciences.
Nanjing Agricultural University.
‡
§
10.1021/bm100289v
 2010 American Chemical Society
Published on Web 06/11/2010

1
702 Biomacromolecules, Vol. 11, No. 7, 2010
Communications
a
Scheme 1. Syntheses of GC Conjugates 1
(C
H
11NO
)
0.53(C22
H
39NO15
)
0.42(C
H
13NO
)0.05: C, 46.69; H, 6.94; N,
6
4
8
5
4
.26. Found: C, 46.53; H, 7.05; N, 4.18.
Hemagglutination Inhibition (HAI) Assay. Hemagglutination
5
,6
assays were performed according to a previously reported method.
2
S. suis was grown overnight at 37 °C in 5% CO incubator. The bacteria
were harvested by centrifugation (5000 × g, 15 min, 4 °C) and washed
twice with phosphate-buffered saline (PBS, pH 7.2). The hemagglu-
tination activity was titrated and the lowest bacterial density causing
agglutination was used for the inhibition studies. Conjugates 1a-f,
conjugate 10, and compound 6 were dissolved in PBS at the concentra-
w
tion of 1.0 mg/mL. Conjugates 1g-i, chitosan (M 300 kDa) were
dissolved in acidic PBS (pH 4.5, adjusted with 1 N HCl) at the same
concentrations. The above solutions were serially diluted (1:10 for the
first time, and then 1:2). PBS (pH 7.2 and 4.5, respectively) was used
as the blank. For inhibition assays, a volume of 25 mL of each dilution
was mixed with an equal volume of bacteria suspension. After
incubation at room temperature for 5 min, a suspension of 4% human
erythrocytes in PBS (50 mL) was added. The hemagglutinations and
minimum inhibitory concentrations (MIC) were recorded.
Surface Plasmon Resonance (SPR) Analysis. The binding of
4
conjugate 1c with BSI-B lectin was analyzed by a BIAcore biosensor
system based on the SPR technique. Conjugate 1c was covalently
immobilized by a standard amine-coupling method on a sensor chip
(
CM-5, research grade) that was coated with carboxymethyl dextran.
A solution of BSI-B lectin at the concentration of 0-0.114 mg/mL
0-1000 nM) in running buffer (PBS, pH 7.2) was injected over the
immobilized conjugate 1c at a flow rate of 20 µL/min for 4 min
association phase). During the dissociation phase, the sensor surface
was exposed to running buffer at the flow rate of 20 µL/min. Conjugate
0 and chitosan (M 300 kDa, DDA 95%) were used as the negative
controls. The affinity constant (K ) of the interactions were calculated
by a standard BIA evaluation software (version 3.1).
Measurements. The M and PDI (M /M ) of starting chitosans and
4
(
(
1
w
d
w
w
n
all conjugates were measured by GPC (CTO-20A, Shimadzu Co., Ltd.)
equipped with a multiangle laser light scattering detector (DAWN
1
HELEOS-II, Wyatt Co., Ltd.) in NaOAc buffer (pH 4.5). The H and
1
3
C NMR spectra were recorded on a Bruker DRX 400 spectrometer
at 400 and 100 MHz, respectively. The DDA of starting chitosans was
1
determined by the corresponding H NMR spectrum. ESIMS and
HRESIMS data were recorded on a Mariner ESI-TOF spectrometer
(ABI Co., Ltd.). Elemental analysis was performed with a Flash EA
1112 instrument (ThermoQuest Co., Ltd.). Surface plasmon resonance
a
Reagents and conditions: (i) BzCl, pyridine, -20 °C, 65%; (ii) AgOTf,
SnCl
2
, CH
2
Cl
2
-Et
2
3
O, 0 °C, 53%; (iii) MeONa, MeOH, and then Na/NH ,
MeOH, -78 °C, two steps, 56%; (iv) O
3
, Me
2
S, -78 °C, quantitatively;
, AcOH (aq. 5 wt %
solution), rt, 65-82%. Degree of substitution (DS) was determined by
(SPR) analysis was performed by a BIAcore biosensor system (Biacore
(
v) chitosan (see Experimental Section), NaCNBH
3
b
3000, Biacore Co., Ltd.).
1
c
the corresponding H NMR spectrum. Degree of polymerization (DP) was
estimated from the M of starting chitosans.
w
Results and Discussion
2
(
4 h at room temperature and then dialyzed with a membrane tube
MWCO: 14 kDa). After filtration to remove the precipitate, lyophiliza-
tion of the filtrate gave conjugate 1 as a white amorphous solid
65-82% yield). The quantity of reaction agents, yield, structural
Syntheses commenced with the assembly of galabiose deriva-
tive 7 having an alkyl linker terminated by an aldehyde group
Scheme 1), which was designed for attaching galabiose unit
(
(
onto the amino groups of chitosan. Selective benzoylation of
parameter of the products (GC conjugates 1a-i), and the element
analysis data were summarized in Supporting Information (Table S1
and Table S2). H NMR of 1c (400 MHz, D
12
13
galactoside 2 according to a published procedure afforded
14
3
in a 65% yield. Glycosidation of galactosyl fluoride 4 with
1
2
O): δ 4.99 (d, 1H, J )
acceptor 3 provided the exclusively R-linked disaccharide 6 after
two steps of deprotection reactions. TLC indicated ozonolysis
of the C-C double bond of 6 proceeded quantitatively to
provide aldehyde 7, which was directly employed for the next
coupling reaction with chitosan without further purification.
Reductive N-alkylation of chitosan with 7 was conducted
smoothly in an aqueous AcOH solution in the presence of
3
.6 Hz, Gal
Gal H-1), 4.41 (t, 1H, J ) 6.4 Hz, Gal
.22-2.74 (brm, 5.4H, GlcN H-2 and NHCH
COCH ), 1.70 (br, 4H, linker CH ). Other GC conjugates gave similar
H NMR data.
R
H-1), 4.67 (br, GlcNAc H-1), 4.50 (d, 1H, J ) 7.6 Hz,
H-5), 4.16-3.54 (m, 21.6H),
), 2.09 (br, 0.28H,
ꢀ
R
3
2
3
2
1
Synthesis of Lactose-Chitosan Conjugate (10). Conjugate 10 was
prepared with a similar procedure as described for GC conjugate 1.
1
1
NaCNBH to afford the GC conjugates 1a-i in 65-82% yield.
3
Ozonolysis of compound 8 (20 mg, 48.8 µmol) yielded aldehyde 9,
which reacted with chitosan (4.2 mg, 25.7 µmol, M 300 kDa, DDA
5%), affording 7 mg (22.1 µmol) of conjugate 10 (42% DS, 83%
The degree of substitution (DS) of the galabiose branch in GC
w
conjugate was adjusted by the molar ratio of aldehyde 7 to the
amino groups of chitosan (see Supporting Information). The
maximum DS was obtained at 56% by using 2 equiv 7. A large
excess of 7, up to 4 equiv, has been tested and found to
have no effect on further increasing the DS value, likely due to
9
1
yield). H NMR (400 MHz, D
J ) 8.0 Hz, Gal H-1), 4.44 (d, 1H, J ) 7.6 Hz, Glc H-1), 3.98-3.47
m, 18.5H), 3.28-2.91 (brm, 6.8H, GlcN H-2 and NHCH ), 2.06 (br,
.35H, COCH ), 1.69 (br, 4H, linker CH ). Anal. Calcd for
2
O): δ 4.90 (br, GlcN H-1), 4.49 (d, 1H,
ꢀ
(
0
2
3
2

Communications
Biomacromolecules, Vol. 11, No. 7, 2010 1703
a
Scheme 2. Syntheses of Lac-Chitosan Conjugate 10
Table 1. Inhibitory Effect of GC Conjugates on the
Hemagglutination of Human Erythrocytes Caused by S. suis
HA9801 Strain
a
sample
DP
307
DS (%)
MIC (ng/mL)
1
1
1
1
1
1
1
1
1
6
1
a
56
52
53
54
54
25
14
8
5 (7.1 nM)
3 (4.1 nM)
b
c
d
e
f
613
1839
6131
33722
1839
1839
1839
1839
1.25 (1.7 nM)
2.5 (3.4 nM)
2.5 (3.5 nM)
2.0 (1.8 nM)
25 (15.3 nM)
5000 (1970 nM)
b
b
g
h
b
c
i
3
NA
5500 (13410 nM)
c
0
1839
1839
42
NA
NA
b
,
d
c
chitosan
a
MIC (minimum inhibitory concentration): ng/mL of sample (nM of
b
galabiose moiety). Sample was dissolved in acidic PBS (pH 4.5) that
was tested no influence on the assay (blank). No activity at the
concentration of 1.0 mg/mL or less. The chitosan with DDA of 95% and
c
d
w
M of 300 kDa was used.
sured by a GPC system was generally at the same level with
that estimated from the formula weight (F ) and DP of chitosans
w
(
F
w
× DP), and the PDIs had approximately the same range as
a
Reagents and conditions: (i) O
3
, Me
2
S, -78 °C, quantitatively; (ii)
, AcOH (aq. 5 wt %), rt.
those of starting chitosans as well (data not shown), suggesting
that little change had occurred in the molecular weight of
chitosan backbones during the synthetic process. However,
attempts for the further characterization by 13C NMR spectros-
copy has failed because of the rigid chitosan backbone and
increased macromolecule size of the conjugates.
chitosan (DDA 95%, M
w
300 kDa), NaCNBH
3
1
5
Inhibitory effect of GC conjugates on the adhesion of S. suis
to human erythrocytes (expressing galabiose-terminated gly-
5
colipids on the surface ) was evaluated by HAI assay. As the
results in Table 1 show, potent inhibition of the conjugates 1a-g
was observed at low nanomolar concentrations. However, the
MIC values were strongly dependent on two structural variants
of the conjugates, that is, the DP of chitosan backbone and the
DS of galabiose branch. Conjugates 1a-e with the similar DS
(
52-56%) but different DP showed the highest inhibitory effect
at the DP of 1839 (1c, MIC ) 1.7 nM), whereas the smaller
1a, 1b) and larger (1d, 1e) conjugates were relatively less
(
effective. Umemura et al. have demonstrated that the scaffold
length of the multivalent inhibitor is a significant factor in
control of their antiadhesion activity through a molecular
Figure 1. 1H NMR spectra of (a) GC conjugate 1c and (b) Lac-
chitosan conjugate 10 in D O at 300 K. Abbreviations: Gal , R-linked
Gal in 1c; Gal , ꢀ-linked Gal in 1c or in 10.
4
d
2
R
simulation study. Here the HIA assay results also supported
the assumption that polymer size was of particular importance
for the access and binding of bacterial cells to the conjugates,
and 1c, with a moderate molecular length, would be the most
favorable one for the S. suis bindings among the conjugates
tested. The result that larger conjugates (1d, 1e) showed a same
activity might indicate a critical point of polymer size existing
for the bacterial preference; otherwise, the conformation of
conjugates such as the distribution and availability of galabiose
branches would also affect the inhibitions as demonstrated by
Umemura et al. In addition, no clear correlation between the
molecular weight distributions and inhibitions has been found
by a comparison of PDI and MIC values of the conjugate (data
not shown). On the other hand, the inhibitory activity decreased
in accordance with the DS value, as illustrated by conjugates
ꢀ
the increased steric hindrance of the conjugate. For negative
control of the bioevaluation of GC conjugates, a lactose (Lac,
Galꢀ1-4Glc) branched chitosan derivative, Lac-chitosan con-
11
jugate 10, was also synthesized from compound 8 in a similar
procedure (Scheme 2). In contrast to water-insoluble chitosan,
conjugates 1a-f and 10 showed good solubility in neutral water
(
solubility g 1.0 mg/mL, PBS, pH 7.2) owing to the highly
incorporated hydrophilic sugar branches, but conjugates 1g-i
with the lower DS value were less soluble.
Characterizations of the conjugates were carried out by H
1
NMR spectroscopy, CHN elemental, and GPC analyses. The
1
structural compositions were verified by H NMR assignments
7
of several baseline-separated signals of the galabiose (or lactose)
moiety, alkyl linker, and acetyl group of chitosan backbone, as
typically illustrated by the spectra of conjugate 1c and 10 in
Figure 1. The DS values determined on the basis of integral
ratio of the branch-derived peaks to chitosan backbone acetyl
peaks were in good agreement with the CHN elemental analyses
1c and 1f-h, due to the multivalency effect. Monovalent
galabioside 6 showed a very weak activity at the MIC of 13410
nM. Conjugate 1i had no activity, even at the concentration of
1.0 mg/mL, probably due to the fact that the large number of
chitosan backbone (extremely low DS) hampered the access
of the galabiose moiety to S. suis cells. In addition, no inhibition
of hemagglutination was observed for conjugate 10 and chitosan,
(
w
see Supporting Information). The M of all conjugates mea-

1
704 Biomacromolecules, Vol. 11, No. 7, 2010
Communications
biocompatible and cheaply available chitosan as the scaffold
that enables not only the large-scale preparation but also the
improved drug safety. The preliminary bioevaluation of GC
conjugates revealed that 1c completely inhibited the S. suis-
induced hemagglutination at a concentration of 1.7 nM, which
6
b
was comparable with the most potent inhibitor known by far
octavalent galabioside with poly(amidoamine) dendrimer scaf-
fold, MIC ) 2.4 nM]. SPR study also revealed that 1c was of
high affinity with the BSI-B lectin (K ) 39.6 nM). Currently,
[
4
d
more extensive and in-depth bioevaluations of the GC conjugate
is being pursued, and efforts are ongoing to develop these
promising antiadhesion agents into the drug for S. suis infection.
Acknowledgment. This work was supported by grants from
MOST (973 Program 2006CB504400), NSFC (30770486), CAS
(
KSCX2-YW-G-032 and KSCX2-YW-R-178), and State Key
Laboratory of Microbial Resource, Institute of Microbiology,
CAS.
Figure 2. Sensorgram for the binding of GC conjugate 1c with BSI-
lectin. Immobilized ligand: 1c on the SPR sensor chip; injected
analyte: BSI-B lectin at various concentrations in PBS buffer
pH 7.2).
B
4
Supporting Information Available. Experimental details;
NMR and elemental analysis data; and NMR and mass spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
4
(
proving that the inhibitions were derived from interactions of
S. suis with the galabiose moieties of the conjugates.
References and Notes
The proposed molecular mechanism of inhibition of multi-
valent galabiose derivatives is the interactions with adhesins
present on S. suis surface. Because galabiose-specific adhesins
(
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(
(
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4
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(
(
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4
lectin with conjugate 1c was detected in a dose-
(
d
)
4
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(
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2
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(
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2
(
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BM100289V