A Highly Sensitive Fluorogenic Probe for Imaging Glycoproteins and Mucine Activity in Live Cells in the Near-Infrared Region

Article abstract of DOI:10.1002/chem.201800790

A novel fluorescent molecular probe is reported, which is able to detect glycoproteins, especially mucins, with high sensitivity and with a turn-on response along with a large Stokes shift (>130 nm), within the biologically active window. The probe contains an aminotricarbocyanine as the fluorescent reporter with a linked benzoboroxole as the recognition unit, which operates through a dynamic covalent reaction between the boronic hemiester residue of the receptor and cis-diols of the analyte. The superior selectivity of the probe is displayed by the labeling of mucins present in Calu-3 cells. The new benzoboroxole fluorescent derivative gathers together key properties to make it a highly rated molecular probe: specificity, excellent solubility in water, and off–on near infrared emission. This probe is expected to be an excellent tool for imaging intracellular mucin to evaluate mucus-related diseases as well as a sensing strategy towards glycosylated structures with a high potential for theranostics approaches in biological samples.

Full text of DOI:10.1002/chem.201800790

A Journal of
Accepted Article
Title: Novel Highly Sensitive Fluorogenic Probe for Imaging
Glycoproteins and Mucine Activity in Live Cells in the Near
Infrared Region
Authors: Cecilia Samaniego Lopez, Jimena Hebe Martínez, María
Laura Uhrig, Federico Coluccio Leskow, and Carla Cecilia
Spagnuolo
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To be cited as: Chem. Eur. J. 10.1002/chem.201800790
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10.1002/chem.201800790
Chemistry - A European Journal
Novel Highly Sensitive Fluorogenic Probe for Imaging
Glycoproteins and Mucine Activity in Live Cells in the Near
Infrared Region
Cecilia Samaniego Lopez,[a]§ Jimena Hebe Martínez,[b]§ María Laura Uhrig,[a] Federico Coluccio
Leskow[b] and Carla Cecilia Spagnuolo*[a]
[a] Departamento de Química Orgánica-CIHIDECAR-CONICET, Facultad de Ciencias Exactas
y Naturales, Universidad de Buenos Aires, Int. Guiraldes 2160, 1428, Ciudad de Buenos Aires,
Argentina.
[b] IQUIBICEN-CONICET, Departamento de Química Biológica, Facultad de Ciencias Exactas
y Naturales, Universidad de Buenos Aires, Int. Guiraldes 2160, 1428, Ciudad de Buenos Aires,
Argentina.
§Authors CSL and JHM contributed equally to this work
*corresponding author’s email address: carlacs@qo.fcen.uba.ar
Abstract
We introduce a novel fluorescent molecular probe able to detect glycoproteins, especially
mucins, with high sensitivity and with a turn-on response along with a large Stokes shift (>130
nm), within the biologically active window. The probe contains an aminotricarbocyanine as the
fluorescent reporter with a linked benzoboroxole as the recognition unit, which operates through
a dynamic covalent reaction between the boronic hemiester residue of the receptor and cis-diols
of the analyte. The superior selectivity of the probe is displayed by the labeling of mucins
present in Calu-3 cells. The new benzoboroxole fluorescent derivative gathers together key
properties to meet high rated molecular probes: specificity, excellent solubility in water and off-
on near infrared emission. The probe we provide is expected to be an excellent tool for imaging
intracellular mucin to evaluate mucus related diseases as well as a sensing strategy towards
glycosylated structures with a high potential for theranostics approaches in biological samples.
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10.1002/chem.201800790
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A large amount of research has been made by chemists over the lasts decades to provide
fluorescent molecular probes for the specific and non-specific labeling of biomolecules escorting
the rapid development of imaging techniques by means of fluorescence, from spectroscopy to
nano/microscopy. Among the possible labeling targets present in a biomolecule structure to
attach a reporter molecule, carbohydrate residues have been an elusive tag in probe design.
This is due mainly to the structural similarity between carbohydrate monomers within a
glycostructure which can be seen as a bunch of OH groups whose subtle structural and
stereochemical differences make difficult a selective recognition process. In addition, in
aqueous media, water molecules represent the main competitor of sugars for a given probe. In
Nature, especially in mammals, only six carbohydrate monomers accounts for a diversity of ca.
1012 different oligosaccharides. Despite this discouraging fact, the challenge of carbohydrate
recognition has been taken by different groups in the last three decades motorized by the
fundamental role of saccharides and their conjugates in major biological processes. Following
mainly supramolecular chemistry strategies to address this point, Davis´s group intensively
developed a biomimetic approach based on non-covalent interactions.[1] On the other hand, a
dynamic covalent strategy arising from the reversible reaction between boronic acids and cis-
diols to give boronate complexes in a reversible way was also explored by different
researchers.[2] Our group is particularly interested in this dynamic recognition event to achieve
carbohydrate recognition in aqueous media focusing in the development of new molecular
fluorescent probes for glycostructures, named boronolectins. Recently, we reported a novel
benzoboronic acid fluorescent sensor with excellent performance for the bulk detection of
glycoproteins in vitro and in live cells.[3] However, this benzoboronic acid probe requires a
membrane permeation step and the affinity is not optimized unless a large concentration of
carbohydrates is offered to the probe.
In order to overcome these limitations (high pKa, low affinity, need of permeation pre-treatment)
we designed a near-infrared (NIR) fluorescent probe that contains benzoboroxole (also named
benzoxaborole) as the recognition unit. We find essential to focus in the development of NIR
fluorophores (650-900 nm) to preclude photodamage of biological samples, to accomplish deep
tissue penetration and high signal-to-noise ratios by minimizing background autofluorescence.
Since the work of Hall et al.[4] benzoboroxole became an improved receptor for sugars showing
high potential for different applications, from saccharide detection in vitro[5], materials science[6]
to medicinal chemistry due to its bioactivity against different pathogens.[6-7] Our unique
fluorescent boronolectin probe named Cy-Bx (7) is presented in this work as a powerful dye to
label mucins in live cells with high sensitivity and selectivity. Cy-Bx achieved some of the high
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rated requirements for a molecular probe to be used in biological systems: excellent water
solubility, NIR fluorescence emission and fluorogenic response.
The receptor 5 was synthesized starting from benzoboroxole according to Scheme 1, by
standard synthetic methods (section 2, Supporting Information). Compound 5 possess a primary
amino terminal group, suitable to perform the radical nucleophilic aromatic substitution reaction
SRN1 with chlorotricarbocyanine 6 at the meso position[8]. We chose compound 4 to explore the
performance of the boronolectin in vitro. Similar to benzoboroxole (pKa 7.3), receptor 4 has a
pKa of 6.9 that matches the physiological range. These values were determined
spectroscopically and are in accordance with those determined by other methodologies[4a]
(Figure S6). Benzoboroxole is more acidic than benzoboronic acids (pKa values in the range 9-
10) and showed to have better affinity for pyranoses than for furanoses in neutral water,
favouring the rehybrydization that follows the formation of the boronate complex.
Moreover, the fact that receptor 4 is slightly more acidic than benzoboroxole correlates with the
observed affinity for tested sugars. Figure 1 shows the direct change in the emission of 4 at 325
nm upon binding of common monosaccharides (Figure S9). In general terms, it follows the
affinity tendency of benzoboroxole (Figure S9, Table S1) with the highest Ka values computed
for fructose and sorbitol (Ka 811 and 637 M-1 respectively) and moderate binding to glucose
(177 M-1), mannose (28 M-1), galactose (58 M-1) and fucose (44 M-1).
The experiments were carried out in buffered solution (PBS, 0.1 M) at pH 7.4 without the need
of using co-solvent. The boronolectin receptor (4) was also studied for the recognition of sugar
related structures like adenosine triphosphate (ATP) and adenosine monophosphate (AMP)
because of their high abundance in biological samples (vide infra). The affinity of 4 to these
analytes was computed to be significantly higher than common monosaccharides (Fig. 1, Table
S1). A cooperative effect for the binding involving the phosphate groups is probably the main
reason for the observed boost in affinity.
Moving forward the synthesis of the fluorescent boronolectin Cy-Bx (7), BOC-deprotected
receptor 5 was reacted with chlorotricarbocyanine 6 in mild conditions as previously described.[3,
9]
Cy-Bx was obtained as a deep blue solid after chromatographic purification and it was
completely characterized by spectrosocopic methods (Figures S3 and S4). In methanol, Cy-Bx
shows a maximum at 650 nm (16724 M-1) in the absorbance spectrum and at 780 nm in the
exc 650 nm). These parameters remain practically unchanged in
the quantum yield in aqueous medium is frequently observed in these cyanine dyes, due to the
formation of aggregates. Cy-Bx has a large Stokes shift (>130 nm) as expected for
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aminotricarbocyanines, an important optical feature for imaging fluorescent probes, as the
interference from excitation light is minimized and the filter settings in the microscopic set up is
optimized. It is worth to note that unlike our previous probe[3] Cy-Bx is not completely quenched
in water, a fact probably related to the presence of the succinic-ethylendiamine linker between
the fluorophore and the recognition unit, that contributes in reducing the formation of typical
aggregates. Besides, the presence of amide groups confers an electronic configuration not
suitable to intramolecular quenching processes such as electronic transfer.
The calculated pKa value for Cy-Bx by spectroscopic methods is 8.5 (Figure S7), more basic
than that of the receptor 4. This can be attributed to the presence of the secondary amine arised
after the nucleophilic substitution of chlorine in the meso position of the heptamethine chain. We
studied the behavior of the complexes between Cy-Bx and model monosaccharides within the
pH range 3-12 (Figure S8). In all cases the pKa values of the complexes were more acidic than
the free probe as a result of the binding process and the concomitant change in geometry of the
boron center increasing the amount of boronate species in the complex.
Next we evaluated the performance of Cy-Bx to detect model glycoproteins in vitro (Figure 2).
We chose to evaluate the binding of the probe to mucin MUC-1 and asialofetuin (Afet) because
these glycoproteins are considered reliable candidates as disease biomarkers. Asialofetuin
expresses the oncofetal Thomson Friedenreich antigen (TF, Gal(β1–3)GalNAc), a cell surface
biomarker for colon cancer and others[10] while MUC-1 is a mucine overexpressed in ovarian,
breast, colon cancer among others.[11]
The boronolectin Cy-Bx also binds with good affinity to fetuin, the sialylated glycoform of
asialofetuin (8% sialic acid). In all cases the binding was detectable at the nanomolar level
yielding an off-on response in the near infrared region (780 nm), in buffered medium at pH 7.4
(Figure 2). When compared to the non-glycosylated control protein (streptavidin), an
hypsochromic shift was observed for the labeled proteins of ca. 30 nm along with emission
enhancement for all the glycoproteins (Figure S10) accounting for the binding event.
To study the performance of the novel boronolectin Cy-Bx as an imaging agent, we conducted
labeling assays toward the mucin granules present in airway epithelial Calu-3 cells, which are
related to human lung adenocarcinoma.[12] In goblet cells, mucin production entails several
biological processes. The biosynthesis of glycosylated mucins requires transcription of a MUC
gene to mRNAs that are translated in the rough endoplasmic reticulum (RER) into proteins that
are post-translationally modified in the Golgi complex by glycosyltransferases. Secretory mucins
are stored in granules and released at the apical surface.[13]
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Imaging the secretion pathway of mucin granules and their kinetics in live cells has been an
elusive task due to the lack of suitable dyes for monitoring this dynamic process. Recently,
Shumilov et al. proposed the use of acridine orange at saturating levels to image the mucin
secretion in ephithelial Calu-3 cells by means of total internal reflection fluorescence (TIRF)
technique.[14] They proposed a two color excitation approach in the visible range by varying the
concentration of the dye and making profit of its ability to interplay between monomers and
aggregates of different color. This enabled the visualization of mucins focusing in the post-
secretory swelling kinetics. This report is so far, the most accomplished example when mucin is
the target. We expected our probe to be superior in this regard, since it can be excited and
imaged within the near infrared range excluding autofluorescence interferences and background
signals. Besides, Cy-Bx includes the benzoboroxol as a specific recognition unit for cis-diols
motifs, introducing better selectivity. Moreover, it was demonstrated by Kreda et al that mucin
granules constitutes a major source of nucleotide uptake/storage/release in the secretory
pathway.[15] Given the observed affinity of the receptor to both, nucleotides and mucin (Figure 1)
and mucin (Figure 2) Cy-Bx must be able to achieve both recognition events enhancing the
efficiency of the binding.
Therefore, human lung adenocarcinoma Calu-3 cells were labeled by gently washing with PBS
and incubated at 37ºC for 30 minutes in modified Tyrode’s buffer containing 50 µM of the probe
Cy-Bx (see section 7 in Supplementary Information for cell maintenance and differentiation
details). In a first experiment we performed MUC5AC immunostaining simultaneously with Cy-
Bx labeling in order to verify its specificity (Figure 3). In all experiments the incubation with the
probe did not affect the cell morphology as compared to control cells.
Co-localization plot corroborates Cy-Bx specificity for mucin granules. The coincidence of both
channels is indicated (Figure 3 E). In the same culture, some Calu-3 cells differentiate into
goblet cell phenotype and are able to produce mucin, while others cells have the ability to
polarize in an apical and basal surface.[16] As we mentioned before, mucin synthesis occurs in
RER and afterwards they are modified in the Golgi complex producing perinuclear localization
(Figure 3).
To further analyze secretory cells labeled with our probe, confocal z-stacks were performed. 3D
reconstructions showed granules spread in cytoplasm as well as with membrane labeling. This
result suggest that Cy-Bx not only is able to stain mucin granules but also labels mucin on cell
surface (Figure 4 A and C). Orthogonal view shows that Cy-Bx endures inside the cells as
granules can be observed in different levels without significant degradation of the probe and
preserving a high quality of the signal (Figure 4 B). This is not a minor observation taking into
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consideration that although a fluorogenic response was observed for mucin in cuvette (Figure 2)
the recognition of ATP or AMP in vitro correlates with a quenching process (Figure S11). Given
the mentioned report of Kreda et al accounting for the heavy cargo of ATP and related
molecules co-existing with mucin within granules, we notice that another effect is empowering
the emissive signal. Such enhancing effect must be given by the inherent viscosity of the
granules and mucin itself increasing the fluorescence quantum yield. To assess this fact was
studied the change of the fluorescence intensity of Cy-Bx by monitoring the emission with
viscosity rendering an enhancement of 90% in the signal at 755 nm (Figure S12). Moreover, the
quenched complex of Cy-Bx with ATP showed an increase in intensity with higher amounts of
glycerol in solution (Figure S13).
The images also allowed us to discriminate different morphologies of the granules in our cellular
model. It is well known that mature mucin secretory granules are large, with a mean diameter of
1 μm.[17] Therefore, size variation correlates with the stage of maturation. Figure 5 shows
different fields of a same culture. Large and small mucin granules are visible in secretory cells.
In summary, we accomplished the synthesis and application of a novel near infrared molecular
probe Cy-Bx with high sensitivity for glycoproteins by combining a benzoboroxole fragment as
the recognition moiety for carbohydrates with a tricarbocyanine fluorophore as the reporter.
Cy-Bx showed a fluorogenic response to the presence of model glycoproteins in cuvette with
negligible non-specific binding. By means of a simple labeling protocol, the probe shows high
specificity for mucin by imaging granules in Calu-3 cells, demonstrated by co-localization
analysis in comparison to a specific antibody.
The versatility of the chemical modification of the tricarbocyanine structure over other NIR dyes
is suitable for designing improved probes, including orthogonal functionalization. Having
achieved higher demands for a fluorescent molecular probe regarding NIR emission, large
Stokes shift and water solubility, we foresee Cy-Bx as an excellent bioanalytical probe for
carbohydrate recognition, filling a vacancy in the fluorescence imaging tool-box.
Acknowledgements
This work was supported by Universidad de Buenos Aires (UBACYT 20020130200070BA),
CONICET (PIP 112 201301 00027) and ANPCYT (2015-2766). C.S.L. and J.H.M. are PhD and
post-doctoral fellows of CONICET, respectively. M.L.U., F.C.L. and C.C.S.are established
research fellows of CONICET.
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Scheme 1. Synthesis of the new probe Cy-Bx (7). i) HNO3, -45ºC, 84%; ii) H2, Pd/C, THF, 7h,
90%; iii) succinic anhydride, EtOH, 75ºC, 10h, 80%; iv) DCC/NHS, EDA-BOC, TEA, DMF, 62%;
v)TFA, DCM; vi) DMF, TEA, 12h, 60%
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300
250
200
150
100
50
A
C
E
G
I
K
M
O
0
-4.5
-4.0
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
Log Csugar
Figure 1. Binding of receptor 4 (3.10-4 M) to model monosaccharides and nucleotides in buffer
PBS 0.1 M pH 7.4, monitored by the change in the emission at 325 nm (exc 250 nm).
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1000
800
600
400
200
Fet
AFet
MUC-1
SAv
-5
-4
-3
-2
-1
log Cprotein
1.0
0.5
0.0
t
c
Fe
Afet
Mu
SAV
Cy-Bx
Figure 2. Fluorogenic response of boronolectin Cy-Bx 2.7 M to model glycoproteins in buffer
PBS 0.01 M, pH 7.4, exc 650 nm.
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Figure 3. Cy-Bx labeled mucin granules. Calu-3 cells mucin granules were labeled with Cy-Bx
and MUC5AC immunostaining (A and B). Merge of confocal microscopy images of the
differential interference contrast (DIC) and Cy-Bx (C). Merge of Cy-Bx and MUC5AC. Yellow
line points out pixels to co-localization analysis (D). Fluorescence intensity versus distance
shows of Cy-Bx and MUC5AC co-localization. White arrows mark coincident intensity among
red and green channel; light blue arrow marks minimal fluorescence intensity of both cannels.
Yellow arrow shows co-localization of red and green channels in another cell region (E). Images
were taken with an objective Oil 60X PLAPON NA=1.42. Co-localization was analyzed
employing FIJI (ImageJ) software.
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Figure 4. Cy-Bx labeled granules in cytoplasm and secreted mucin. 3D-reconstruction of
confocal Z-stacks shows a Calu-3 cell with mucin granules throughout the entire cytoplasm.
(A)Yellow line was drawn for the subsequent analysis of the granules. White arrows points out
mucin granules and possibly secreted mucin localized on cellular membrane. (B) Orthogonal
view shows granules localization inside the cell. (C) merge among DIC and Cy-Bx labeled cells.
3D reconstruction and orthogonal view were performed employing FIJI (ImageJ) software.
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Figure 5. Cy-Bx labeled mucin granules with different morphologies. Merge of DIC and Cy-Bx
images show different stages of mucin granules in the same cell culture (A, B and C). Images
analysis was performed employing FIJI (ImageJ) software.
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Table of contents
Boronolectin based Near-IR fluorescent probe: a benzoboroxol-tricarbocyanine based probe
with large Stokes shift, fluorogenic response and high selectivity for glycoproteins was
developed. The performance of the dye was demonstrated in live epithelial cells by imaging
mucin granules. It was possible to detect differences in morphology and localization of mucin
within the cells. The novel probe is expected to be a useful tool for bioanalytical imaging of
glycostructures.
Keywords
boronolectin; cyanine; glycoprotein; fluorescent probes; dyes/pigments
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