A tetravalent sialic acid-coated tetraphenylethene luminogen with aggregation-induced emission characteristics: design, synthesis and application for sialidase activity assay, high-throughput screening of sialidase inhibitors and diagnosis of bacterial vaginosis

Article abstract of DOI:10.1039/c8cc06300a

We report a turn-on tetravalent sialic acid-coated tetraphenylethene luminogen (TPE4S) with excellent hydrophilicity, good stability, high sensitivity and unique selectivity towards sialidases, and the maximum fluorescence enhancement was ～40 fold. More importantly, TPE4S was successfully utilized for the screening of sialidase inhibitors and diagnosis of bacterial vaginosis.

Full text of DOI:10.1039/c8cc06300a

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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A tetravalent sialic acid-coated tetraphenylethene luminogen
with aggregation-induced emission characteristics: design,
synthesis and application for sialidase activity assay, high-
throughput screening of sialidase inhibitors and diagnosis of
bacterial vaginosis
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Guang-jian Liu,†a Beihan Wang,†b Yuan Zhang,a Guo-wen Xing,a,* Xiaoli Yang,b, * and Shu Wang c
We report
a
turn-on tetravalent sialic acid-coated high-throughput drugs screening and rapid diagnosis of various
tetraphenylethene
luminogen
(TPE4S)
with
excellent diseases associated with sialidase-producing bacteria to
hydrophilicity, good stability, high sensitivity and unique alleviate the patients’ discomfort and reduce serious
selectivity towards sialidases, and the maximum fluorescence complications. To date, a few protocols were reported for the
enhancement was ~40 folds. More importantly, TPE4S was sialidase activity assay with mono-sialic acid derived
successfully utilized for screening of sialidase inhibitors and chromophores as substrates.6 In the previous work, we have
diagnosis of bacterial vaginosis.
developed a continuous fluorometric assay for sialidase
activity using water-soluble conjugated polyelectrolytes
through the “on-off” fluorescent response.7 However, most of
these methods are limited in their functions. For example,
generally, 2’-(4-methylumbelliferyl)-α-D-N-acetyl-neuraminic
acid was only used for screening of sialidase inhibitors and 2’-
Sialidases (or neuraminidases, EC 3.2.1.18), which cleave the
glycosidic bond in sialo-glycoconjugates specifically and
efficiently, are widespread in virus, bacteria and mammalian
cells.1 In most cases, sialidase’s activity was associated with
the pathogenicity of many pathogenic microorganisms.2 For
example, hemagglutinin and sialidase of influenza viruses are
two key virulence factors in the processes of viruses invasion
and spread of new virions, and some licensed existing drugs
for influenza infection, such as oseltamivir and zanamivir, are
considered to function by targeting and inhibiting the sialidase
of influenza viruses.3 In addition, it appears likely that the
sialidase secreted by Streptococcus is responsible for neonatal
and infant meningitis through desialylating mucins at the
meningeal surface.2 Similarly, Vibrio cholerae sialidase could
hydrolyze the polysaccharides on the surface of intestinal
mucosal cells to produce the receptor of cholera enterotoxin,
which facilitated the penetration of toxins and caused
disease.4 Moreover, as one of the specific enzymes produced
by bacterial vaginal infections, sialidase has been used as an
important marker of bacterial vaginosis (BV) for clinical
diagnosis.5
(5-bromo-4-chloroindol-3-yl)-α-D-N-acetyl-neuraminic
acid
was only used for diagnosis of bacterial vaginosis.6 It is highly
desired to develop new sialidase detection agents with
multiple functions.
Over the last decade, aggregation-induced emission (AIE)
materials have attracted great attention due to the unique
photophysical properties, which are unlike the commonly used
fluorophores with aggregation-caused quenching (ACQ)
effect.8 Various AIEgens have been extensively applied for
biosensing and imaging in living systems based on
coordination binding, electrostatic interactions or chemical
reactions between AIEgens and analytes. The restriction of
intramolecular motion caused by aggregation, high viscosity,
cryogenic temperature or “locked” structures is the main
working mechanism of this special fluorescent effect.9
sialic acid
Sialidase
TPE
TPE
Based on the sialidase activity assay, it is critical to realize
sialic acid
TPE4A
TPE4S
Insoluble, aggregation, Fluorescence on
40-fold fluorescence enhancement
Water soluble, Fluorescence of f
OH
OH
CO2
O(CH2
OH
HO
HO
AcHN
HO(H2C)3
NHAc
OH
(CH2)3OH
N
N
N
(CH2)3O
O
)
3
O
N
N
N
N
N
N
N
N
HO
CO2
HO
N
N
N
N
N
N
OH
HO
OH
OH
N
CO2
O(CH2
NHAc
OH
HO
AcHN
N
N
N
N
N
O
(CH2)3O
N
HO(H2C)3
(CH2)3OH
O
)
3
TPE4A
TPE4S
CO2
HO
HO
Scheme 1 Design of TPE4S for specific detection of sialidase
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Considering that most classic AIE cores, such as
tetraphenylethene, are hydrophobic, we envisioned that the
incorporation or subsequent removal of hydrophilic sialic acids
would significantly change the solubility of these AIEgens,
further resulting in the changes in the existence states and
fluorescence intensities of these molecules. More specifically,
as shown in Scheme 1, once sialic acids were covalently linked
to the propeller-shaped tetraphenylethene luminogen, the
multiple copies of sialic acid-functionalized TPE molecule
(TPE4S) with excellent solubility could give no emission due to
the radiationless relaxation. Upon treated with sialidase to
uncage sialic acid moieties, the emission of TPE4A will be
rejuvenated by the synergistic effects of water solubility
decrease and aggregated nanoparticles formation.
Figure 1. (a) Time dependent relative fluorescence intensity of
TPE4S (20 μM) in PBS (6.7 mM) upon addition of 10 or 50 mU
‧
mL-1 sialidase. (b) Emission spectra and (c) fluorescence
responses of 20 μM TPE4S to various concentrations of added
sialidase. The spectra were obtained in PBS (pH 7.1, 6.7 mM) 1
h after the addition of sialidase. Each point in (c) represents
mean ± SD of triplicate determinations. (λex = 380 nm). (d) Plot
of relative fluorescence intensity (I/I0) at 510 nm versus pH
values in the range pH 4.74 – 7.95. I and I0 are the
corresponding fluorescence intensity at 510 nm of TPE4S at
each pH value in the presence and absence of sialidase from
Clostridium perfringens respectively.
To explore our attractive ideas, we synthesized TPE4S
according to Scheme S1. Firstly, TPE was functionalized with
alkynyl groups to obtain a model dendrimer scaffold (TPEA).
Then, TPE4S-Ac was obtained by click coupling TPEA with the
azido containing sialic acid derivative Sia-N3, which was
synthesized from N-acetyl-5-N,4-O-carbonyl-protected p-
toluene 2-thio-sialoside (Sia-donor) based on the preactivation
strategy with (p-Tol)2SO/Tf2O as promoters developed by our
group.10 Finally, treatment of TPE4S-Ac with sodium
methoxide in methanol followed by saponification of the
methyl ester groups using LiOH in THF-H2O solution afforded
the desired sialic acids-coated TPE probe (TPE4S). The
structure of TPE4S was confirmed by NMR spectroscopy and
HRMS spectra.
mU‧
mL-1, which allows the convenient quantitative detection
of sialidase over this concentration range (Fig. S3a-3b). By
comparison, we found that longer incubation time could
improve the detection sensitivity of TPE4S. Particularly
interesting, when the incubation time was extended to 24 h,
very low concentration sialidase could still hydrolyze TPE4S
effectively, and produced satisfactory fluorescence
enhancement (Fig. S3c). If we define the detection limit as the
sialidase concentration at which 3-fold fluorescence intensity
increased (I/I0) could be measured, the detection limit was
Then the fluorescence spectra of 20 μM TPE4S in phosphate
buffer solution (pH 7.1) were determined with time varied
from 10 minutes to 24 hours. As shown in Fig. S1a, the solution
of TPE4S was weakly emissive to give stable and minimal
background signals due to the good solubility of TPE4S in
buffer solution. Meanwhile, the fluorescence spectrum nearly
parallel to the abscissa also verified the nonemissive nature of
sialidase from Clostridium perfringens (Fig. S1b). Fig. 1a depicts
the fluorescence kinetic curves of TPE4S upon incubation with
~0.1 mU‧
mL-1 for sialidase upon incubation with TPE4S for 24 h
(Fig. S3d), indicating the sensitivity of TPE4S for sialidase
activity assay.
10 or 50 mU‧
mL-1 sidalidase. In principle, higher concentration
of sialidase could result in faster cleavage of sialoside and
The fluorescence intensity of TPE4S before/after incubation
with sialidase at various pH values was also investigated. As
shown in Fig. S4a, the fluorescence of TPE4S remained silent
over the physiological pH range, and pH values hardly had
effects on the fluorescence intensity of its hydrolytic product
(Fig. S4b). More importantly, similar fluorescence
enhancement could be obtained upon addition of sialidase
from Clostridium perfringens to TPE4S solutions at pH 4.74-
7.95 (Fig. 1d). In addition, the reactivity of TPE4S toward Vibrio
faster fluorescence enhancement. For sialidase from
Clostridium perfringens at 50 mU
intensity of the solution of TPE4S could reach a plateau (
fold) in 20 min (Fig. S2). However, in the case of 10 mU
mL-1, it
took 90 min to reach plateau. The kinetic data implied that
‧
mL-1, the fluorescence
40-
∼
‧
∼
the fluorescence response could be utilized for the rapid
detection of sialidase. To achieve the quantitative detection of
sialidase, a fixed incubation time was chosen in the following
sialidase assays.
cholerae sialidase (50 mU ‧
mL-1) was also evaluated by
measuring the fluorescence responses in PBS buffer solution
and sodium acetate buffer solution. There was negligible
fluorescence at 510 nm for TPE4S at pH 5.0 and 7.1 (Fig. S5).
However, after treatment TPE4S with Vibrio cholerae sialidase,
remarkable fluorescence enhancement could be detected.
These results indicated that TPE4S was applicable to detect
sialidases under the physiological pH range.
As shown in Fig. 1b and 1c, TPE4S behaved as a turn-on
fluorescent sensor under physiological conditions when
incubated with sialidase at 37°C for 1 h. The fluorescent
emissions of the solution were monotonically increased as the
concentration of sialidase increased from 0 to 50 mU‧
mL-1.
Similarly, the fluorescence intensity at 510 nm of the solution
of TPE4S after incubation with sialidase for 2 h was also
detected and the maximum fluorescence intensity increased
substantially as the concentration increased from 0 to 6
The selectivity of TPE4S toward sialidase over other
biological analytes was then examined. Almost negligible
change in fluorescence signal was observed in the presence of
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various common sugars (Fig. S6), and there were also no cell of ITC instruments containing relatively low amount of
changes in fluorescence intensities upon the addition of 2.0 Clostridium perfringens sialidase, plot of rate vs TPE4S
equiv. of common metal ions in the buffer solution (pH 7.1) concentration was plotted affording the complete kinetic
(Fig. S7). These results showed that the excellent selectivity of curve (Fig. 2c). The apparent KM value of TPE4S was calculated
TPE4S toward sialidase over other prevalent bio-molecules, to be 24.8 μM, which was much lower than that of mono-sialic
which is required to achieve efficient sialidase assay in acid derived chromophores, such as 2’-(5-bromo-4-
practical applications.
chloroindol-3-yl)-α-D-N-acetyl-neuraminic acid (about 890
For gaining more insight into the detection mechanism, the μM).6b The same ITC experiments with multiple injections of
specific enzyme-triggered sialic acid-free TPE4A formation TPE4S to Vibrio cholerae sialidase was also performed (Fig. S9),
(Scheme 1) was verified by ESI-MS spectra and dynamic light further indicating that TPE4S was a good substrate (KM, 77.5
scattering (DLS) analyses. For the solution of TPE4S with μM) for sialidases. These results demonstrated that our sugar-
adequate sialidase, a high abundance of the ion at 855.3836 coated dendrimer TPE4S had an increased affinity to sialidase
corresponding to TPE4A in the MS spectra (Fig. S8) was owing to its multivalency, i.e. one probe with four sialic acid
obtained, indicating that TPE4S could indeed be hydrolyzed by units, which could lead to higher local concentrations for the
breaking the glycosidic bond upon sialidase-catalyzed reaction enzyme-catalyzed reactions.
to release the hydrophobic TPE moiety (TPE4A) and four sialic
Discovery of new inhibitors of viral and bacterial sialidases
acids. The hydrolysate TPE4A accumulated to form aggregates plays a paramount role in preventing the spread of severe
with average hydrodynamic diameter about 1200 nm as diseases and pandemics associated with sialidase, which
determined by DLS due to the decreased solubility in aqueous makes it necessary to develop high-throughput inhibitors
solution (Fig. 2a and 2b). Once the luminophores aggregate, screening strategies.12 Since we had confirmed that TPE4S
molecules are located in the immediate vicinity impeding the could be applied for detection of sialidase activity, its
intramolecular rotations, and the adjoining luminophores suitability for the screening of sialidase inhibitors was
experience intense fluorescence enhancement exhibiting examined based on the fluorescence intensity changes. The
typical AIE phenomena.
inhibition efficiency of well-known inhibitors, N-acetyl-2,3-
As the rate of enzyme substrate decomposition reaction is dehydro-2-deoxyneuraminic acid (Neu5Ac2en), zanamivir and
proportional to the reactive heat power output, further oseltamivir acid (the active ingredients of precursor drug
enzyme kinetics analysis to obtain Michaelis-Menten constants oseltamivir), was re-evaluated by our probe using a 96-well
(KM) of TPE4S in the enzyme-catalyzed reaction was carried out plate assay. Different concentrations of inhibitors were
by isothermal titration calorimetric (ITC) technique.11 By prepared and added into the 96-well plate with the total
analyzing the complete thermodynamic profile obtained from volume in each well 200 μL, and the final concentrations of
multiple injections of 1.5 mM TPE4S solution into the sample TPE4S and sialidase from Clostridium perfringens were 20 μM
and 10 mU‧
mL-1 respectively. The control groups were set to
be that without inhibitors and the blank groups contained
TPE4S buffer solution only. Fluorescence readings were taken
at 510 nm on a fluorescence plate reader after 1 h shaking at
37°C. From the plots in Fig. 2d, when TPE4S was incubated
with the sialidase pretreated with Neu5Ac2en (the
commercially available sialidase inhibitor) for
fluorescence intensity at 510 nm decreased substantially as
the concentration of Neu5Ac2en increased from 7.5 M to 1
mM. The IC50 value of Neu5Ac2en was determined to be 73
1 h, the
ꢀ
ꢀ
M, which was very close to the previous result (67 ꢀM)
reported by Linhardt.13 Likewise, in the case of the licensed
drugs, oseltamivir (acid) and zanamivir, which were much
Figure 2. Hydrodynamic diameters distribution of (a) TPE4S weaker inhibitors of bacterial sialidase than viral sialidase, the
and (b) its hydrolytic product TPE4A at the concentration of 20 inhibition efficiency determined by our assay were also well
ꢀM in PBS (pH 7.1, 6.7 mM) as determined by dynamic laser consistent to the earlier results.7,12 Detailedly, the IC50 value of
scattering. (c) Calorimetric determination of enzyme kinetic oseltamivir acid was estimated to be about 98
ꢀM and the
parameters for the hydrolysis of TPE4S by sialidase from inhibition efficiency of 1 mM zanamivir was found to be 0.22.
Clostridium perfringens at 37°C. (d) Evaluation of inhibition These results in turn corroborate the reliability of our method
efficiency of potential inhibitors on sialidase from Clostridium for sialidase activity assay.
perfringens using
efficiency was expressed using the relative fluorescence caused by the imbalance of vaginal ecosystem due to the
intensity (IC )/(IC I0), where and IC are the fluorescence overgrowth of sialidase-producing bacterial pathogens like
a
96ꢁwell plate assay. The inhibition
Bacterial vaginosis (BV) is a common gynecologic disease
ꢁ
I
ꢁ
I
intensity at 510 nm of mixtures with and without potential Gardnerella Vaginalis. Generally, BV is considered to be
inhibitors respectively and I0 is the fluorescence background of associated with endometritis, pelvic inflammatory disease and
20 ꢀM TPE4S itself. (λem = 510 nm).
adverse pregnancy outcome.5 Recently, the chromogenic
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Table 1 Diagnostic results obtained by BVBlue test and acids. TPE4S exhibited good hydrophilicity and adequate
investigated TPE4S fluorescence test.
Determined by TPE4S fluorescence test
sensitivity towards sialidase, and the maximum fluorescence
enhancement was ~40 folds when sialidase triggered the
deglycosylation. Determination of KM constant, ESI-MS and DLS
experiments confirmed that TPE4S could be hydrolyzed by
sialidase to release hydrophobic TPE4A to undergo rapid
accumulation to report sialidase activity under physiological
conditions. The very low KM value (24.8-77.5 μM) of TPE4S in
the sialidase-catalyzed reaction indicated an increased affinity
of TPE4S with sialidase compared with the previous
monosaccharide derived chromogenic substrate. Most
importantly, TPE4S was utilized for the screening of sialidase
inhibitors successfully and also turned out to be a potential
and promising diagnostic agent for the bacterial vaginosis
caused by sialidase-producing anaerobic bacteria.
No. (%) of samples
Weak
Determined by
BVBlue test (No. of
samples)
Strong
Positive
(I/I0 > 10)
3 (2.7)
Normal
positive
(5< I/I0 ≤ 10)
(0< I/I0 ≤ 5)
Normala(110)
101(91.8)
3 (7.5)
6(5.5)
BV (40)
7 (17.5)
30 (75.0)
a Cleaning degree of vagina: I and II.
BVBlue test for diagnosis of BV, which was based on the
detection of elevated sialidase produced by anaerobic
pathogens in vaginal fluid samples qualitatively, has been
found to be reliable compared with the conventional methods
such as Amsel criteria and Nugent scoring system.5 As the
presence of sialidase is currently considered as a key indicator
of BV in the clinical examination, TPE4S was then investigated
to show the feasibility as a powerful diagnostic tool for the
high throughput fluorescence-guided diagnosis of BV.
The project was financially supported by the National
Natural Science Foundation of China (21272027).
Conflicts of interest
There are no conflicts to declare.
A total of 150 reproductive-aged women presented in
gynaecological clinics were recruited into the study, which
were divided into two groups according to the diagnosis
results by BVBlue test and microscopic observations for vaginal
cleanliness determinations (healthy group: 110; BV group: 40).
The vaginal fluid specimens collected from each patient were
also diagnosed by incubation with TPE4S at 37°C. Based on the
relative fluorescence intensities (I/I0) monitored at 510 nm of
experimental groups (I) and control group (I0) in which only 20
μM TPE4S was added, we graded the samples as normal (grade
1, 0< I/I0 ≤ 5), sialidase weak positive (grade 2, 5< I/I0 ≤ 10), and
sialidase strong positive (grade 3, I/I0 > 10). The results
obtained by both the BVBlue test and our TPE4S method are
shown in Table 1. 91.8% of participants having negative
BVBlue tests result showed no positive reaction with our
TPE4S fluorescence test, and only three (2.7%) samples from
this group were strong positive by our method. Besides, the
sialidase-positive samples determined by BVBlue test also
demonstrated a high proportion of weak/strong positive
results (92.5%), with three of forty patients (7.5%) being in the
normal grade by our test. The sensitivity and specificity of our
fluorescence-guided method compared with the diagnosis
results by BVBlue test were found to be 92.5% and 91.8%
respectively. In addition, leukocyte esterase, another routine
examination item in gynecological examination, could not lead
to the changes of the fluorescence intensity of TPE4S (Fig.
S10). Meanwhile, the kappa coefficient (0.805, P<0.001)
indicates good agreement between our new method and the
Notes and references
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our method should give more accurate classification and
quantification of the severity of BV based on the relative
fluorescence intensity (I/I0), indicating the enormous clinical
potential of TPE4S as a new simple diagnostic tool for facile
diagnosis of BV.
,
12 M. Richter, L. Schumann, E. Walther, A. Hoffmann, H. Braun,
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In summary, we have developed a “smart” sugar–coated
turn-on AIEgen (TPE4S) with multivalency of sialic acids.
Dendrimer TPE4S was equipped via “click” coupling reaction
between an alkynyl terminated TPE and azide-annexed sialic
4 | J. Name., 2012, 00, 1-3
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A tetravalent sialic acid-coated tetraphenylethene
luminogen with aggregation-induced emission
characteristics: design, synthesis and application for
sialidase activity assay, high-throughput screening of
sialidase inhibitors and diagnosis of bacterial vaginosis
Guang-jian Liu, Beihan Wang, Yuan Zhang, Guo-wen Xing,*
Xiaoli Yang, * and Shu Wang
A sialic acids-coated tetraphenylethene luminogen with excellent hydrophilicity
was reported for sialidase activity assay and diagnosis of bacterial vaginosis.