A water-soluble AIE-active polyvalent glycocluster: Design, synthesis and studies on carbohydrate-lectin interactions for visualization of Siglec distributions in living cell membranes

Article abstract of DOI:10.1039/c9cc05008f

In this work, we designed and synthesized an aggregation-induced emission (AIE)-active tetraphenylethene-decorated pseudo-trisialic acid (TPE3S) and validated its high affinity for Siglecs using microscale thermophoresis techniques. TPE3S was a unique binding-on fluorescent trivalent sialocluster which was successfully utilized for the visualization of Siglecs expressed on the surface of mammalian cells.

Full text of DOI:10.1039/c9cc05008f

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Water-soluble AIE-active polyvalent glycocluster: design,
synthesis and the studies on carbohydrate-lectin interactions for
visualization of Siglecs distributions on living cell membranes
Received 00th January 20xx,
Accepted 00th January 20xx
Guang-jian Liu,a Yuan Zhang,a Lingyun Zhou,b Li-yan Jia,a Guohua Jiang,c Guo-wen Xing*a and Shu
Wangb
DOI: 10.1039/x0xx00000x
In this work, we designed and synthesized an aggregation-induced such as facile functionalization and strong photobleaching
emission (AIE)-active tetraphenylethene-decorated pseudo-trisialic resistance in the sensing field and the AIE mechanism is the
acid (TPE3S) and validated its high affinity for Siglecs using restriction of intramolecular motions,16,17 we envisioned that
microscale thermophoresis techniques. TPE3S was
a unique non-fluorescent and water-soluble AIEgens should act as the
binding-on fluorescent trivalent sialocluster which was successfully “binding-on” probes upon binding with Siglecs to restrict
utilized for the visualization of Siglecs expressed on the surface of intramolecular motions and to realize the visualization of Siglecs
mammalian cells.
distribution pattern on cell membranes with low background
signals. Besides, since high affinity and high specificity of lectins
binding could be achieved as a result of the polyvalency of the
interactions,10,13 we designed a trivalent sialocluster (TPE3S)
decorated by tetraphenylethene, which is a typical AIE-active
fluorophore (Scheme 1). The binding of TPE3S to available
recombinant human Siglecs in the form of Siglec–Fc chimeras
(Fc = fragment crystallizable region of an antibody), like Siglec-
2, -3, -5, was assessed by microscale thermophoresis (MST)
which provides the means for interaction analyses with very low
consumption of samples.18
Contrastive experiments carried out with chemically modified
sialic acid glycocluster mimetics (TPE1S and TPE4S),
demonstrating that the high Siglec-binding affinity of TPE3S was
highly dependent on the multivalency of sialic acid moiety and
the flexible structure in TPE3S (Scheme 1 and 2), which may
trigger tremendous conformational polymorphism to fit into
Siglec topography better. In addition, TPE3S was successfully
used to bind to an array of (secreted) Siglecs for the
concomitant assessment of Siglec distribution on PC-12 cell and
HeLa cell surfaces. The tetravalent sialic acids-coated molecule
TPE4S which had lower affinity for Siglecs but high sensitivity for
sialidases, was used for the imaging of cellular sialidase activity
(Scheme S1). Our current work provided a “binding-on” strategy
to visualize Siglecs without involving the use of antibodies that
are relatively expensive and unstable, and would also be benefit
to maximize the signal-to-noise ratio.
Sialic acid binding immunoglobulin-like lectins (Siglecs) which
contain sialoside binding N-terminal domains are an important
family of lectins predominantly present on the surface of tumor
cells, hematopoietic cells and immune cells.1,2 All Siglecs are
single-pass transmembrane proteins and are heavily involved in
immunoregulation and other physiological functions by
recognizing and reversibly binding sialosaccharides.3,4 A wide
range of potential Siglecs-targeting drug candidates, such as
epratuzumab, inotuzumab ozogamycin, and gemtuzumab
ozogamicin etc., have provided opportunities for innovative
therapeutic strategies for many kinds of immunological
disorders or cancers.5-8 Based on the previous research,9-13
rational modifications of sialic acid glycoside framework and
preparations of sialic acid glycoclusters are two effective ways
to lead to the discovery of specific ligands for Siglecs.
Until now, there are some molecular biology tools such as
fluorescent antibodies, dye-tagged glycoprobes, biotinylated
probes, to locate Siglecs on living cell membranes.14-15 However,
all these dye-labeled molecular tools belongs to the “always-
on” fluorescent probes with high background signals, which not
only result in the low sensitivity but also need the washing steps
before imaging (Scheme S1a). Developing facile and effective
“binding-on” probes to replace previous “always-on” probes for
detecting and visualizing the glycan-binding receptors on the
cell surface is of great value but has not been investigated.
Considering that the aggregation-induced emission (AIE)
materials have been illustrated to possess many advantages,
Initially, to develop fluorescence-based glycoprobes with AIE
characteristics to visualize the Siglecs distribution pattern on
living cell membranes, the concept of modular design was used
to construct Siglec-specific trisaccharide mimic of sialic acid
(TPE3S). TPE3S includes three parts: i) the hydrophobic
tetraphenylethylene (TPE) fluorophore, which not only permits
TPE3S to be detected when it interacts with Siglecs, but is also
conducive to increase the interactions of TPE3S and cell
membrane by its hydrophobic characteristics;19 ii) the multiple
a. College of Chemistry, Beijing Normal University, Beijing, 100875, China.
b. Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China.
c. Analysis & Testing Center, Beijing Normal University, Beijing, 100875, China.
Electronic Supplementary Information (ESI) available: Experimental procedures for
TPE3S and TPE1S; structural characterization data for TPE3S, TPE1S and in-
termediates; supplementary fluorescent spectra and cells images. See
DOI: 10.1039/x0xx00000x
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Scheme 1 Schematic of tetraphenylethene-decorated sialic acid derivative TPE3S structure, measuring and imaging.
solubility in PBS buffer. Whereas, an increased fluorescence of
TPE3S with large Stokes shift value in the mixture of PBS and
glycerin was observed since higher liquid viscosity restricted the
rotation of the phenyl rings of TPE3S molecules, indicating that
the maintenance of AIE activity of TPE fluorophore after
incorporating multiple sialic acids into it. The good solubility of
TPE3S in aqueous solution and the typical AIE activity of TPE3S
should endow the probe with the advantages of low
background noise and wash-free property over other analytical
methods when used for visualizing Siglecs in vivo.
Scheme 2 Structure of TPE4S and TPE1S.
For
analyzing
the
binding
behavior
between
tetraphenylethene-decorated sialic acid glycocluster TPE3S and
Siglecs, MST assays18 were performed at different
concentration ratios of the binding partners. Firstly, Siglec-2
was fluorescently labelled and kept at a constant concentration
and TPE3S was titrated until a saturation of all binding sites
were obtained. From the obtained data in Fig. 1a, the
equilibrium dissociation constant (Kd) was determined to be
about 14.2 μM, which was much smaller than that between
natural sialosides and Siglecs (0.1–3 mM).1,22 Absolute binding
affinity between TPE3S and Siglec-3 was also determined using
MST measurements. Fig. 1b indicated that the Kd was
determined to be 96.2 μM. Similarly, we then quantified the
dissociation constant of TPE3S with Siglec-5 in PBS buffer and
an affinity of 10.6 ± 3.3 μM was obtained (Fig. 1c). These results
showed that TPE3S with flexible structures could bind directly
with the CD33-related Siglecs (Siglec-3, Siglec-5) and the
conserved Siglecs (Siglec-2) successfully.
To determine whether the measured MST response
represented specific binding, bovine serum albumin (BSA) and
mannoside-binding protein Concanavalin A (Con A), were
chosen to evaluate the binding with TPE3S. The titration results
show that no significant binding response could be observed
with BSA or Con A upon addition of TPE3S (Fig. S2 and 1d), and
non-specific binding of TPE3S with Siglecs was excluded.
sialic acid moieties which play crucial roles in carbohydrate-
lectin binding interactions; iii) the connecting backbone,
pentaerythritol with flexible structure, which gives multiple
sialic acid moieties adequate flexibility to tune their spatial
arrangement to exactly target different Siglecs. The synthesis of
TPE3S was based on a versatile, converging, three-stage
modular synthetic protocol shown as in Scheme S2. Firstly,
azide-annexed sialic acid derivative Sia–N3 was obtained using
the preactivation strategy with (p-Tol)2SO/Tf2O as glycosylation
promoters.20,21 Then, azide-functionalized tetraphenylethene
fluorophore was synthesized from 4,4'-dimethylbenzophenone
and 4-bromobenzophenone in two steps, followed by coupling
with perpropargylated pentaerythritol to yield TPE3 in
moderate yields. TPE3S-Ac was prepared using the copper-
catalyzed azide-alkyne Huisgen cycloaddition in the presence of
CuSO4 and sodium ascorbate. Finally, treatment of peracyl
protected probe TPE3S-Ac with sodium methoxide in methanol
followed by saponification of the methyl ester groups using
LiOH afforded the fully deprotected product TPE3S in 70% yield.
The structure of TPE3S was confirmed by NMR spectroscopy
and HRMS spectra. In addition, the contrastive compound
TPE1S was synthesized similarly (Scheme S3).
As shown in Fig. S1, sugar-bearing TPE3S exhibited greatly
quenched fluorescence in aqueous solution due to its good
Fig. 1 Thermophoretic analysis of interactions between lectins and ligands in PBS. To determine the affinities of binding reactions, a titration series of concentrations of sialic acid
bearing ligands partners were performed respectively while the fluorescent binding protein partners were kept at a constant concentration. The interactions of (a) Siglec-2, (b) Siglec-
3, (c) Siglec-5, and (d) Con A with TPE3S (green spots), TPE4S (deep red spots) and TPE1S (blue spots) were measured as described in the supporting information.
2 | J. Name., 2012, 00, 1-3
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Next, to further verify whether the carboxyl groups in TPE3S undergo topographical changes to better mVimiewicArticnleaOtunlrinael
DOI: 10.1039/C9CC05008F
molecule could lead to false results in binding assays, sialosides to act as potential ligand to recognize Siglecs, while
contrastive compound TPE1S functionalized with an equal the space distribution of four sialic acid moieties in TPE4S has
numbers of carboxyl groups with TPE3S was used to exclude relatively “fixed” positions. Moreover, since TPE3S can bind
non-specific contributions by electrostatic interactions. with Siglecs more tightly than natural sialosides (0.1–3 mM),1,22
Compared with TPE3S, no signal changes were obtained using it should make TPE3S a powerful tool for Siglecs biosensing.
TPE1S in the MST titrations with Siglec-2, Siglec-3 and Siglec-5,
We then utilized TPE3S as a novel probe to study the Siglecs
respectively (Fig. 1a-c). We further analyzed the interactions distribution patterns on cells based on the fluorescence
between TPE1S and BSA and Con A with MST respectively, and techniques. After TPE3S was incubated with PC-12 cells for 5
found no binding for BSA and Con A (Fig. 1d and S2). These hours, a bright fluorescence mainly on the surface of PC-12 cells
results substantiated the propositions that the presence of was observed by confocal microscopy, whereas no obvious
multiple sialic acid moieties, rather than carboxyl groups, fluorescence could be observed in the culture medium and in
enhanced the observed affinity of small-molecule ligand for cells (Fig. 2). Colocalization experiments with commercial
Siglecs. Also, we can conclude that the weakness of single sialic organic fluorogen (DiD) available for cell membrane monitoring
acid-Siglec interactions may require the attachment of multiple demonstrated that TPE3S mainly accumulated on cell surface in
sialic acid moieties to ligand for an efficient complex formation, a patch-wise distribution. Even after incubation PC-12 cells with
i.e. multivalence glycocluster effect may trigger downstream TPE3S for 24 hours, we found that most TPE3S molecules were
responses that can be not achievable with single sialic acid.
still impermeable to cell membranes and accumulated on cell
To gain a better understanding of the interaction mechanisms surface discretely (Fig. 2). These results indicated that TPE3S
between sialic acid binding membrane-anchored lectins and could specifically interact with Siglecs on the cytoplasmic
potential ligands, similar experiments were also performed with membrane of PC-12 cells to restrict its intramolecular motions
tetrasialic acids-coated tetraphenylethene (TPE4S),26 which has and trigger an efficient “turn-on” fluorescence.
relatively rigid structure features compared with TPE3S. To our
Compared with sialocluster TPE3S, which had relatively
surprise, the dissociation coefficient of TPE4S with Siglec-2 was higher binding affinities for Siglecs, TPE4S featured binding
determined to be Kd = 3.17 ± 0.8 mM, which is 223-fold higher affinities in the millimolar range. To examine the ability of TPE4S
than that between TPE3S and Siglec-2 (Fig. 1a and Table S1). A to image Siglecs, confocal fluorescent imaging was also
binding affinity of 1.29 ± 0.22 mM for Siglec-3 and no binding performed in cultured PC-12 cells. Fig. S3 shows that TPE4S was
for Siglec-5 were further afforded from the MST experiments, not prone to accumulate on cell surface and the intracellular
which were also higher than that of TPE3S under the same fluorescence was detected. Meanwhile, no fluorescence was
conditions (Fig. 1).
observed in the culture media without washing process before
The binding affinities of three sialic acid-hinged molecules imaging. As TPE4S was an excellent probe for sialidase activity
with different lectins are summarized in Table S1. The data determination,23 the light-up fluorescence signals in cells
illustrated that although the ligand affinity for Siglecs was low strongly suggested that TPE4S was cell membrane permeable
at the level of a monovalent carbohydrate–Siglec binding and should be hydrolyzed by the intracellular sialidases
interaction (like TPE1S), glycocluster effect indeed resulted in a specifically to break the glycosidic bond to form hydrophobic
significant higher affinity for Siglecs (like TPE3S). More hydrolytic product. Further colocalization studies (Fig. S4) with
importantly, it appeared unlikely that more sialic acid moieties LysoTracker Red as standard lysosomal markers demonstrated
could lead to higher affinity between low-molecular-weight that the fluorescent molecules was mainly localized in
ligands and Siglecs. The structure of sialylated molecules lysosomes. These results were well consistent with the previous
should have great influence on the affinities for Siglecs. We research results, i.e. sialidases Neu1 and Neu4 expressed in
reasoned that TPE3S could deduce three sialic acid “paws” to mammalian cells mainly existed in lysosomes.24 Therefore, for
the first time, TPE4S was considered as a potential tool with a
visual sensitivity for identification and in situ imaging of cellular
sialidases under physiological conditions.
Furthermore, as a negative control, TPE1S can not bind with
Siglecs and there was only around a 1.5-fold increase in the
fluorescence intensity after immersion of TPE1S with siliadase
(Fig. S5). We found that after treatment of PC-12 cells with
TPE1S, fluorescence images showed much weaker than those
after incubation with TPE3S or TPE4S (Fig. S6), which in turn
reflected the low affinity of TPE1S with Siglecs and the poor
capability of TPE1S for sialidase detection.
Collectively, in some sense, the cellular imaging experiments
explicitly reflected the consistency of the above affinity studies
Fig. 2 Fluorescence confocal images of PC-12 cells incubated with TPE3S and commercial
dyes DiD or LysoTracker Red (DiD is a commercially available membrane probe,
LysoTracker Red is a commercially available lysosomal probe). Left panel is the confocal
carried out on MST. Better than TPE4S and TPE1S, TPE3S
selectively recognized and interacted with Siglecs on the cell
surface due to its higher affinity to sialolectins, resulting in the
image from TPE3S on channel 1 (λex = 405 nm, λem = 445-545 nm); Middle panel is the
confocal image from DiD (λex = 635 nm, λem = 655-755 nm) or LysoTracker Red (λex = 559
nm, λem = 570-620 nm) on channel 2. Scale bars = 10 μm.
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J. Name., 2013, 00, 1-3 | 3
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This work was financially supported by BeVijiienwgArtiNcleaOtunlrinael
DOI: 10.1039/C9CC05008F
Science Foundation (2192025) and Natural Science Foundation
of China (21272027).
Conflicts of interest
There are no conflicts to declare.
Notes and references
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Fig. 3 Fluorescence confocal images of HeLa cells incubated with TPE3S (50 μM) and
commercial dye DiD (DiD is a membrane probe). Left panel is the confocal image from
TPE3S on channel 1 (λex = 405 nm, λem = 440-540 nm); Middle panel is the confocal image
from DiD on channel 2 (λex = 635 nm, λem = 650-750 nm). Scale bars = 10 μm.
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bright fluorescence images of Siglecs due to the AIE
characteristics. Aimed to build a novel platform that could
sensitively image Siglecs and sialidases using TPE3S and TPE4S
respectively, the biocompatibility and toxicity of TPE3S and
TPE4S were evaluated by MTT method. The data indicated that
there were no significant differences in cell viability between
the initial and that after incubation for 24 h, suggesting the low-
toxicity of the sialyl derivatives used in this study (Fig. S7).
To further validate the efficiency of TPE3S for the detection
of Siglecs expressed on the surface of mammalian cells, similar
experiments were subsequently conducted using cervical
cancer HeLa cells. Interestingly, colocalization experiments
performed using DiD as control revealed that TPE3S principally
enriched on the membrane of HeLa cells discretely after
incubation for 5 hours (Fig. 3). Even after incubation with TPE3S
for 24 hours, TPE3S still “shine” on the DiD membrane probe.
The observed intense emission of TPE3S presented on HeLa
cells further indicated that the specific binding between TPE3S
and Siglecs. Therefore, TPE3S could be used to target Siglec-
expressing cells and to visualize Siglecs efficiently.
In conclusion, we described an AIE-active binding-on
fluorescent Siglec-targeting sialocluster (TPE3S) consisting of
multivalent sialic acid moieties. TPE3S was equipped via click
coupling reactions of alkynyl terminated pentaerythritol, azide-
annexed TPE and sialic acids. The specificity of TPE3S toward
Siglec-2, -3 and -5 was determined using MST technique which
was suitable for the analysis of various binders. The contrast
experiments with TPE1S and TPE4S suggested that both the
number of sialic acid moieties and their spatial arrangement
were indispensable for the promising capability of TPE3S
toward Siglecs. Furthermore, TPE3S was used for the
visualization of Siglecs expressed on the surface of PC-12 and
HeLa cells successfully. However, TPE4S with lower affinity for
Siglecs but being high sensitive for sialidase has been shown to
be useful in imaging cellular sialidase with satisfactory
biocompatibility. In addition, in comparison with the antibodies
against Siglecs, TPE3S was a broad-spectrum ligand for most
Siglecs with relatively low specificity and affinity. Further
development of high-affinity ligands for individual Siglec
members by chemical modifications of the sialic acid backbone
is in progress.
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