Multicolor GLUT5-permeable fluorescent probes for fructose transport analysis

Article abstract of DOI:10.1039/c7cc09809j

The specificity of carbohydrate transporters towards their substrates poses a significant challenge for the development of molecular probes to monitor sugar uptake in cells for biochemical and biomedical applications. Herein we report a new set of coumarin-based fluorescent sugar conjugates applicable for the analysis of fructose uptake due to their free passage through the fructose-specific transporter GLUT5. The reported probes cover a broad range of the fluorescence spectrum providing essential tools for the evaluation of fructose transport capacity in live cells.

Full text of DOI:10.1039/c7cc09809j

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DOI: 10.1039/C7CC09809J.
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Multicolor GLUT5-Permeable Fluorescent Probes for Fructose
Transport Analysis
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
b
a
b
a
V. V. Begoyan, Ł. J. Weseliński, S. Xia, J. Fedie, S. Kannan, A. Ferrier, S. Rao, M. Tanasova *
DOI: 10.1039/x0xx00000x
www.rsc.org/
The specificity of carbohydrate transporters towards their Among fluorescent GLUT probes, 7-nitrobenzofurazan (NBD) has
substrates pose a significant challenge for the development of been utilized for GLUT transport analysis in the form of sugar
7
-9
molecular probes to monitor sugar uptake in cells for biochemical conjugates. Among those, fructose and 2,5-anhydro-D-mannitol
and biomedical applications. Herein we report a new set of were accessed as probes to target fructose transport providing a
coumarin-based fluorescent sugar conjugates applicable for precedent for distinguishing GLUT5-expressing vs. GLUT5-deficient
8
, 9
analysis of fructose uptake due to their free passage through the cell lines. Recently, coumarin and resorufin conjugates of glucose
10
fructose-specific transporter GLUT5. The reported probes cover a have been evaluated as probes for glucose GLUTs. The probes
broad range of the fluorescent spectrum providing essential tools showed a limited passage through GLUTs, and accumulated in cells
1
1
for evaluation of fructose transport capacity in live cells.
through non-GLUT-mediated transport. The challenge in passing a
non-natural moiety through GLUTs is also reflected by the loss of
1
2
Facilitative glucose transporters (GLUTs) perform gradient GLUT-mediated uptake for glucose-drug conjugates, with the
dependent influx and efflux of carbohydrates to sustain the position of functionalization and possible change in transporter-
1
nutritional needs for the cell proliferation. Metabolic deregulations probe interaction due to alterations in H-bonding being key
6, 13
in cells induce alterations in cellular GLUT composition, resulting in players.
2-4
overexpression of intrinsic or expression of extrinsic GLUTs.
Herein we report a set of 2,5-anhydro-D-mannitol-coumarin-
Between GLUTs, glucose-transporting GLUT1 gathered a half- based GLUT5-specific probes – ManCous - amenable for the direct
century attention as a target for cancer therapy and diagnostics, analysis of GLUT5 activity in cells and signal reporting over a broad
and other GLUTs started to gather much interest recently because range of the fluorescent spectrum. The specific targeting of GLUT5
3
of their direct relationship with cancer. A particular attention is enables a significant differentiation in imaging of GLUT5-expressing
drawn by fructose-transporting GLUTs and fructose-specific cells vs. GLUT5-deficient cells. The range of fluorescence color
transporter GLUT5 due to the links between fructose uptake and allows a flexibility in conducting evaluation of GLUT5 uptake
5
cancer development, progression, and metastasis. Consequently, efficiency in the presence of fluorescent reporters of other cellular
the fructose transport-targeting probes are of interest as processes, including glucose metabolism, by mismatching probe
biochemical and biomedical tools.
fluorescence excitation and emission.
Analyses of GLUT expression and activity as means to assess the
To access multicolor fluorescent probes compatible with GLUT-
metabolic state of the cell has been approached through mediated uptake, we focused on coumarins as fluorophores. The
development of GLUT-targeting radiolabeled sugar analogs, as well choice was based on their small size and the potential to tune
6
as affinity labels. Among radiolabeled analogs, halogenated sugar fluorescence color through coumarin core functionalization. The
derivatives were accessed to evaluate glucose and fructose passage of 7-aminocoumarin in the form of a glucosamine
1
0
transport efficiency in conjunction with and independently of conjugate through glucose GLUTs has been demonstrated,
6
phosphorylation. Development of fluorescently-labeled GLUT providing basis for testing this fluorophore as a substrate for
probes has been approached to alleviate the practical limitations GLUT5. Thus, as a proof-of-principle, we have investigated the
associated with radiolabeling and to obtain high-affinity probes. uptake of a 7-aminocoumarin as a conjugate of GLUT5-targeting
9
, 14
2
,5-anhydro-D-mannose.
The 7-aminocoumarin (Cou1) was
synthesized from the 7-hydroxycoumarin according to the
1
5
established procedure and conjugated to 2,5-anhydro-D-mannitol
Man) through reductive amination with 2,5-anhydro-2-
carbaldehyde-D-mannitol to produce the first blue-fluorescent D-
(
1
6
mannitol-coumarin conjugate ManCou1 (Fig. 1a).
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With the blue-fluorescent ManCou1 probe in hand, we moved Cou1, highlighting its passive diffusion through the membrane into
8
, 17
into testing its uptake in MCF7 cells known to express GLUT5.
After treating MCF7 cells with various concentrations of ManCou1, observed for higher concentrations of ManCou1 (Fig. S1e),
significant accumulation of the coumarin- induced blue suggesting the involvement of GLUT uptake even at elevated
the cell. The loss of the uptake at low temperature was also
a
fluorescence was observed after a short 10 min incubation (Fig. S1). concentrations.
We found that concentrations ≥20 µM were sufficient to monitor
the probe accumulation through microscopy or with plate reader.
Figure 2. ManCou 1 (20 µM) uptake analysis. a) Confocal Z-stack images
of MCF7 cells treated with ManCou1 for 10 min at 37 °C; b)
Comparative uptake of equimolar ManCou1 and Cou1 at 37 °C and 4 °C;
c) ManCou1 (20 µM) uptake in the presence of 50 mM fructose,
glucose, and glucosamine and 200 µM cytochalasin B. Data collected
with fluorescent plate reader (exc/em 360 nm/430 nm) in 96-well plate
format; d) Comparative analysis of ManCou1 uptake in MCF7 vs. HepG2
cells. Data collected through quantification (ImageJ) of whole-cell
fluorescence (obtained with EVOS optical microscope, exc/em 405
nm/461 nm, 20X objective) after background subtraction. All plots
represents average data from triplicate measurements. Error bars
represent the standard deviation between triplicate experiments.
Figure 1. Structure and spectroscopic properties of ManCou probes. a)
Structures of ManCous 1-14; b) UV-vis spectra for ManCous 1-14; c)
Fluorescence spectra for ManCous 1-14, exc. 385 nm. All spectra taken
for 20 µM ManCou conjugate. *For clarity of representation, the UV
spectrum for ManCou9 is reported for 10 µM concentration.
Considering that ManCou conjugate represent a merge of a
hydrophilic sugar and a hydrophobic fluorophore, we have
investigated the contribution from GLUTs vs. passive diffusion to
the observed cellular accumulation of this probe. The comparative
analysis of the unconjugated 7-aminocoumarin (20 µM, 10 min, 37
We have further used a series of competitive uptake and
inhibition analyses to verify the uptake of ManCou1 through GLUTs
and particularly through GLUT5. We have observed that the uptake
of ManCou1 is effectively inhibited by fructose (Fig. 2c and S2a),
suggesting the probe to be taken through fructose-transporting
GLUT(s). The Kd = 3.1 mM (Fig. S2b) measured for fructose fits
°C) in MCF7 cells showed ~10-fold lower accumulation (Fig. 2b),
highlighting a significant contribution from the sugar moiety to the
observed ManCou uptake. The acquired fluorescence was primarily
from the coumarin association with the cell membrane, as evident
from the Z-stack analysis of Cou1-treated MCF7 cells (Fig. S1c).
While ManCou1 uptake was concentration-dependent, the uptake
levels for the unconjugated coumarin Cou1 were not affected by
concentration (Fig. S1d). The concentration-dependent uptake and
the unambiguous contribution from the sugar moiety to the
enhanced uptake provided basis to consider the GLUT-mediated
transport as a primary uptake mechanism for ManCou1. The GLUT
participation became further evident from the loss of ManCou1
uptake after incubating MCF7 cells at 4 °C (Fig. 2b) - conditions
known to decrease cell metabolism and thereby GLUT-mediated
within the low micromolar binding range established for other
14, 18
GLUT5-proficient cell lines or the isolated protein.
When
19
glucose and glucosamine (specific for GLUT2 ) were used as a
competitive inhibitors, no alterations in ManCou1 uptake were
observed (Fig. 2, c and d) indicating the lack of contribution from
glucose or non-specific fructose GLUTs, including GLUT2. The lack of
ManCou1 inhibition in the presence of cytochalasin B (Fig. 2e) – the
established inhibitor of the uptake through GLUTs 1-4 (IC50 = 2-6
20
µM) but not GLUT5, further ruled out non-specific transport,
highlighting GLUT5-mediated uptake.
The GLUT5-specificity of ManCou1 became further evident from
the lack of the probe uptake in GLUT5-deficient liver carcinoma
8
uptake. In contrast, low temperatures did not impact the uptake of
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HepG2 cells (Fig. 2d). The basal levels of uptake measured for both green and blue filters; ManCou5 was visible under orange/red
HepG2 cells rise from the association of the probe with the filters (Fig. 3) and ManCous 10-14 were visible under the red,
membrane, as no internalisation of the probe is observed in Z-stack yellow, and green filter (Fig. 3). While taken up by MCF7 cells,
analysis (Fig. S3). The observed discrimination shows the feasibility ManCous 2-14 did not enter HepG2 cells suggesting that coumarin
to distinguish between GLUT5-expressing vs. GLUT5-deficient cell modification did not impact their GLUT5-specificity (Fig. S3). The
though fluorescence-based analysis with ManCous. The lack of GLUT5-specificity was also supported by the lack of uptake
inhibition from glucose enabled the use of ManCou1 analysis in inhibition in the presence of glucose, glucosamine, cytochalasin B or
buffer or culture media (Fig. S2f), providing a convenient tool for in complete culture medium (Fig. S2). The inhibitory effect of fructose
vitro cell studies in nutrient-rich conditions.
was strong for ManCous 2, but decreased for EWG-substituted
With the successful delivery of
a blue-fluorescent 7- ManCous.
aminocoumarin into the cell through GLUT5, we moved forward
with testing ManCou analogues to gauge the tolerance of GLUT5 to
coumarin fluorophores and gain access to probes of different
fluorescent color. The substitution at the C4 position of the 7-
aminocoumarin had been shown to impact the fluorescence
2
1
emission. Thus, we constructed a focused library of ManCous (Fig.
a) using readily available coumarin analogues (Cou) 2, 3, and 7,
and extended the library by synthesizing Cous bearing various
1
1
6, 22, 23
electron withdrawing groups at the C4 (C4-EWG),
including
new amides, 2-furyl (Cou13), and 2-pyridyl (Cou14) C4-analogs. We
have completed the library by adding the 6-aminocoumarin (Cou8)
and the C3-substituted analogue of 7-aminocoumarin Cou9 to
assess the impact of alterations in the position of sugar conjugation
on the ManCou uptake.
The Cous 5, 13 and 14 (Fig. 1) were synthesized through
Figure 3. Fluorescence confocal Z-stack images of MCF7 cells treated
with: a) ManCou2; b) and c) ManCou3; d) ManCou4 (blue and green
merge); e) ManCou5; f) ManCou9; g-i) ManCou 11. Blue fluorescence
1
6, 22, 24
Pechmann condensation,
and Cous 10-12 were obtained
1
6
from Cou5 through amidation with the corresponding amine.
Except for ManCou9, all ManCous were obtained through reductive measured at 461 nm; green fluorescence measured at 525 nm; red
fluorescence measured at 585 nm. ManCous were excited with 405 nm
amination of the corresponding Cous with 2,5-anhydro-2-
laser. Yellow color assigned considering the fluorescence maxima for
carbaldehyde-D-mannitol. ManCou9 was obtained through
ManCous 10-12. Images taken with 60X objective after treating cells
EDCI/HOBt-mediated amidation with 1-amino-2,5-anhydro-D-
with 20 µM ManCous over 10 min. Images recorded at the same laser
intensity and exposure time.
mannitol (Scheme S2).
The obtained ManCous cover a broad range of the fluorescence
spectrum (Fig. 1c and Table S1) while maintaining excitation at low
The Z-stack analysis of ManCou-treated MCF7 cells revealed an
wavelength (405 nm). The fluorescence intensity of ManCous varies
apparent impact of coumarin substitution on the cellular
with the substitution position and type, with ManCou13 and
distribution of the probe. Namely, while ManCous 1 and 2 were
ManCou8 showing the highest and the lowest fluorescence
distributed throughout the cell, C4-EWG ManCous and ManCous 8
intensity, respectively. The absolute quantum efficiency for all
and 9 accumulated in the cytosol (Fig. 3). The cytosolic distribution
ManCou conjugates reflects the quenching effect of electron
could be readily delineated through the labelling of the cell nucleus
delocalization from the coumarin scaffold into the electron
and the cell membrane with dyes of contrast fluorescent color (Fig.
withdrawing substituent (Table S2). We have also observed that the
S4). The differences in cellular accumulation of ManCous paralleled
absorption maxima of ManCou conjugates have red-shifted 8-22
the differences in their uptake. Thus, while linear uptake was
nm compared to the unconjugated coumarin (Table S1). In an effort
observed for ManCous 1 and 2 at 1-500 µM concentrations, the
to explain the changes in the absorption, density functional theory
uptake for all other ManCous saturated after 200 µM (Fig. S1a).
was employed. The structure of 7-aminocoumarins and the
Considering that the continuity of the uptake through GLUTs is
corresponding 1-AM conjugates were first geometrically relaxed,
and then single point energy calculations were performed. The
analysis of HOMO and LUMO energies revealed a reduction of the
energy gap upon alkylation of the exocyclic amine (Table S1)
25-27
coupled with phosphorylation,
it is feasible that the differences
in the uptake behaviour between ManCous could be reflecting the
impact of coumarin substitution on phosphorylation of 2,5-
28, 29
anhydro-D-mannitol by cellular kinases.
expectedly contributing to the shift of the
energy.
λmax to the lesser
All saturable ManCous showed Kd in 54-75 µM range (Fig. S5).
Considering that no saturation was observed for ManCou1 uptake
even at elevated 5 mM concentrations, we used fructose: ManCou1
ratio measured to induce 50% ManCou1 uptake inhibition (Fig. S2b)
to estimate 156-fold higher affinity for ManCou1. Overall, the
presence of EWG groups at the coumarin appears to contribute to
the strength of ManCou-GLUT5 interaction as well as uptake rates
Through confocal imaging, we have observed that MCF7 cells
gained fluorescence after treatment with all ManCous except for
ManCous 6 and 7 – both bearing carboxylic acid moieties. The
gained cell fluorescence for ManCous 2, 8 and 9 (blue filter)
highlighted the tolerance of GLUT5 towards positional isomers of
coumarin-mannitol conjugates. ManCous 3 and 4 were visible under
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(
Fig. S5e). This contribution is manifested by the apparent inhibition 2. M. Mueckler and B. Thorens, Mol. Aspects Med., 2013, 34,
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K. Adekola, S. T. Rosen and M. Shanmugam, Curr. Opin. Oncol.,
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of ManCou1 uptake in the presence of ManCou3. Namely,
incubating MCF7 cells with the equimolar mixture of ManCous 1
and 3 resulted in the loss of nuclear accumulation of the blue
fluorescence, a feature characteristic for ManCou1. To explore this
effect further, we have employed GLUT5-targeting green
3
.
2
4
5
.
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A. M. Navale and A. N. Paranjape, Biophys. Rev., 2016, 8, 5-9.
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fluorescent NBDM probe (Kd = 22 µM). After incubating MCF7 cells
6
7
.
.
with the equimolar mixture of ManCou3 and NBDM, a trace of
NBDM-induced green fluorescence was observed inside the cell. In
2
005, 64, 207-215.
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S6a). For ManCou1-NBDM mixture, the uptake of both was
observed, although the total fluorescence intensities were
significantly diminished (Fig. S6b). Overall, the differential effect of
ManCous 1 and 3 on NBDM appear to reflect the differences in the
strength of GLUT5-ManCou interaction. It should be noted that the
M. Namavari and S. S. Gambhir, Bioconjugate Chem., 2007, 18,
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7
(
NBD conjugate of glucose ) was not impacted by any of the
ManCou conjugates (Fig. S6, c and d). This observations further
validates the GLUT5-specificity of ManCou conjugates.
In conclusion, we have shown that fructose-specific transporter
2
319-2333.
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,5-anhydro-D-mannitol (ManCous) includes fluorescent probes
that emit at different parts of the fluorescence spectrum, while
maintaining the same excitation. As GLUT5 reporters, the probes
allow for a visual discrimination between GLUT5-proficient and
GLUT5-deficient cells. The structure-uptake relationship established
with ManCou analogs revealed that the presence of a carboxylate
moiety compromises GLUT5-mediated uptake, while ester, amide,
1
448-1454.
1
1
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nuclear and membrane dyes. Furthermore, variations of fluorescent
colors within the ManCou library provides an opportunity for co-
analysis of GLUT5 and other GLUTs (or other cellular targets).
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2
This research reported in this publication was supported by
the Michigan Technological University and Portage Health
Foundation under Award Number R01563 to M. Tanasova.
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