Generalizable synthesis of bioresponsive near-infrared fluorescent probes: Sulfonated heptamethine cyanine prototype for imaging cell hypoxia

Article abstract of DOI:10.1039/d1ob00426c

Continued advancement in bioresponsive fluorescence imaging requires new classes of activatable fluorescent probes that emit near-infrared fluorescence with wavelengths above 740 nm. Heptamethine cyanine dyes (Cy7) have suitable fluorescence properties bu

Full text of DOI:10.1039/d1ob00426c

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Generalizable synthesis of bioresponsive
near-infrared fluorescent probes: sulfonated
heptamethine cyanine prototype for imaging
cell hypoxia†
Cite this: Org. Biomol. Chem., 2021,
19, 4100
Kirk M. Atkinson,
Bradley D. Smith
Janeala J. Morsby,
*
Sai Shradha Reddy Kommidi
and
Continued advancement in bioresponsive fluorescence imaging requires new classes of activatable fluor-
escent probes that emit near-infrared fluorescence with wavelengths above 740 nm. Heptamethine
cyanine dyes (Cy7) have suitable fluorescence properties but it is challenging to create activatable probes
because Cy7 dyes have a propensity for self-aggregation and fluorescence quenching. A new synthetic
strategy is employed to create a generalizable class of hydrophilic bioresponsive near-infrared fluorescent
probes with appended sulfonates that provide excellent physiochemical properties. A prototype version is
triggered by nitroreductase enzyme to undergo self-immolative cleavage with a large enhancement in
fluorescence signal at 780 nm and the probe enables microscopic imaging of cell hypoxia with “turn on”
fluorescence. Near-infrared fluorescence imaging of hypoxia is potentially useful in many different areas
of biomedical research and clinical treatment.
Received 4th March 2021,
Accepted 13th April 2021
DOI: 10.1039/d1ob00426c
rsc.li/obc
Cyanine heptamethine (Cy7) fluorophores are the most
common dye platform for creating NIR fluorescent imaging
Introduction
Fluorescent near-infrared (NIR) dyes are very attractive for bio- and sensing probes with absorption/emission wavelengths
logical imaging and sensing because there is maximal pene- above 740 nm.7 A range of different photophysical mecha-
tration of light through thick biological samples including the nisms have been used to create bioresponsive Cy7
skin and tissue of living subjects, and also minimal scattering fluorophores.7,8 Progression towards clinical translation
of light which enhances image contrast.1 The design challenge requires the field to produce next-generation Cy7 fluorescent
is to convert a fluorescent NIR dye into a molecular probe that probes with an optimized combination of photophysical,
operates effectively in complex biological media. Basically, chemical and pharmacokinetic properties. In many cases, this
there are two major classes of fluorescent NIR molecular means new and improved Cy7 fluorophore structures with
probes; those that are “always on” or those that are “biorespon- appropriate water solubilizing groups that prevent undesired
sive”, that is, they “turn on” fluorescence after a chemical molecular association processes that degrade bioresponsive
transformation is induced by the biological environment.2,3 signaling such as self-aggregation and accumulation in off-
This latter class of bioresponsive probes has the attractive attri- target biological locations. In this regard, a large fraction of
bute that strong fluorescence signal is observed only at a bioresponsive Cy7 probes are derived synthetically by substitut-
specific bioresponsive region of space or point in time, and ing the C4′–Cl in precursor Cy7-Cl (Scheme 1) with a group
the background signal beyond the bioresponsive region is very that has a nucleophilic O or N atom.9 This synthetic chemistry
low, which ensures high image contrast.4,5 The potential works reasonably well with organic-soluble versions of Cy7-Cl,
benefit of this bioresponsive approach is driving ongoing but it is rarely reported with water-soluble variants that have
research efforts to develop new classes of activatable NIR fluo- appended sulfonate and quaternary ammonium groups.10 The
rescent probes.6
inability to easily prepare hydrophilic, bioresponsive Cy7
probes with substituted heptamethine chains, tunable phar-
macokinetics and high “turn on” fluorescence is currently a
significant impediment to further progress. A good illustration
of this synthetic deficiency is the published literature on small
molecule Cy7 probes that respond to the hypoxia-related
enzyme nitroreductase (discussed further below).11 Very few
Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University
of Notre Dame, IN 46556, USA. E-mail: smith.115@nd.edu
†Electronic supplementary information (ESI) available: Synthesis and character-
ization, supplementary figures and references. See DOI: 10.1039/d1ob00426c
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Table 1 Photophysical properties of dyes 1–7 in methanola
abs
em
λ
λ
ε
Quantum
yield (%)
Fluorescence
brightnessc
max
max
Dye
(nm)
(nm)
(M−1 cm−1
)
1
2
3
4
5
6
749
779
754
785
782
792
774
826
781
824
814
821
242 000d
150 000d
83 400
51 860
153 350
51 510
13.2d
0.6d
31 944
900
9132
84
347
83
11.0b
0.16b
0.23b
0.16b
a All measurements at room temperature. b Relative to 1 in methanol,
error is 10%. c Fluorescence brightness = ε × quantum yield, error is
15%. d Data from ref. 5.
Scheme 1 Chemical structures.
reported examples are sulfonated Cy7 structures and they only
exhibit modest levels of “turn on” fluorescence.10,12,13
Recently, Štacková and coworkers reported a potential way
to overcome this synthetic limitation by developing a new
method to make Cy7 dyes by ring opening of Zincke salts.5,14
We subsequently used this innovative synthetic chemistry to
design a water-soluble Cy7 fluorophore for bioconjugation and
fabrication of “always on” fluorescent NIR molecular
probes.15,16 We now report that the Zincke salt synthetic meth-
odology can be used to create a new class of water-soluble, sul-
fonated bioresponsive NIR Cy7 fluorescent probes with excel-
lent physiochemical properties and very high “turn on” fluo-
rescence. We demonstrate this advance in fluorescent NIR
probe design by developing a prototype probe that is triggered
by nitroreductase enzyme.
Scheme 2 Conceptual picture of bioresponsive NIR fluorescent Cy7
probe based on a self-immolation reaction (1,6-elimination). The reac-
tive 1,4-quinone methide by-product is scavenged by water to generate
a benzyl alcohol derivative.
Results and discussion
Štacková and coworkers evaluated a series of analogous Cy7
fluorophores with different functional groups located along
the heptamethine chain.14 They found that in organic solvent
the Cy7 fluorophore 1 with a C4′-carboxylic acid group was group. Chemical activation of the trigger group by a stoichio-
blue-shifted by 30 nm and 64 times brighter than its corres- metric or enzyme catalyzed reaction reveals an unstable
ponding C4′-methyl ester derivative 2. Intrigued by this obser- phenol or aniline derivative that spontaneously fragments to
vation we prepared the water-soluble sulfonated derivative 3 produce the corresponding C4′-carboxylic acid with enhanced
with C4′-carboxylic acid and the corresponding sulfonated C4′- fluorescence. A by-product of the self-immolation process is a
methyl ester version 4 and observed a very similar large differ- reactive 1,4-quinone methide derivative that is scavenged by
ence in fluorescence brightness (Table 1). This type of func- water to generate a benzyl alcohol.
tional group dependence is unusual; there are very few litera-
We tested this probe design hypothesis by developing two
ture fluorophores that are weakly fluorescent as a carboxylic prototype NIR fluorescent probes that indicate the over-
ester but highly fluorescent as the corresponding carboxylic expression of nitroreductase enzyme associated with cell
acid and none that emit in the NIR.17–21 We reasoned that the hypoxia. In short, cells and tissue respond to low oxygen con-
fluorogenic effect could be combined with a self-immolative ditions (hypoxia) by biosynthesizing reductase enzymes such
cleavage mechanism, such as a 1,6-elimination, to produce a as nitroreductase.24 Several fluorescent NIR probes with
new class of “turn-on” NIR fluorescent probes.22,23 The generic 4-nitrobenzyl groups are known to be triggered by nitroreduc-
probe structure in Scheme 2 is comprised of a Cy7 fluorophore tase to undergo self-immolative fragmentation and fluo-
with a C4′-benzyl ester group that has an appended trigger rescence enhancement.11 With this precedence in mind, we
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prepared the analogous Cy7 probes 5 and 6, each with a C4′- of all the Cy7 compounds in Scheme 1. As expected, the
nitrobenzyl ester group. The synthesis of these two Cy7 dyes, absorption bands for hydrophobic 1 and 5 in PBS are broad
by ring opening of Zincke salts, is described in the ESI.† An due to dye self-aggregation (confirmed by Dynamic Light
important strategic advantage with this synthetic method is Scattering, Fig. S14†). In contrast the absorption bands for sul-
that the water-solubilizing groups (sulfonates in the case of 6) fonated versions 3 and 6 in PBS are narrower indicating less
are introduced in one step after the trigger group (in this case, self-aggregation. Cuvettes studies showed that treatment of
a 4-nitrobenzyl ester) has been installed on the polymethine separate samples of 5 and 6 with nitroreductase enzyme and
chain.5 The key reaction is the catalyzed transimination the necessary cofactor NADH produced the expected changes
process shown in Scheme 3, which occurs at room temperature in absorption and emission (Fig. 1). That is, a 30 nm blue
and tolerates a wide range of functional groups. We anticipate shifted absorption band appeared corresponding to the pro-
that this synthetic sequence will be a convenient way to make duction of carboxylates 1 and 3 (probe cleavage confirmed by
a wide range of new water-soluble bioresponsive Cy7 probes HPLC analysis, see Fig. S17†) along with a concomitant large
with the general molecular design shown in Scheme 2.
increase in fluorescence emission at ∼780 nm. The kinetic pro-
In Fig. 1 and Fig. S3–S9† are the absorption and emission files in Fig. 1 also indicate that the enzymatic cleavage of 6 was
spectra for separate solutions of the four compounds 1, 3, 5, faster than the cleavage of 5. Presumably this kinetic differ-
and 6 and listed in Tables 1 and S1† are the spectral properties ence is due to the probe aggregation state, with the enzyme
able to gain faster access to the less aggregated probe 6.‡ A
control experiment showed that the Cy7 derivatives 2 and 4,
each with a C4′-methyl ester group does not respond to nitrore-
ductase (Fig. S20†), consistent with enzyme catalyzed
reduction of the nitro group as the chemical process that trig-
gers the fluorescence response. Furthermore, there was no
fluorescence enhancement of 5 or 6 when NADH, the necess-
ary nitroreductase cofactor, was omitted from the sample, or
when the known nitroreductase inhibitor, dicoumarol, was
added to the sample (Fig. S19†). A series of cuvette experi-
ments that mixed separate samples of 5 or 6 with various
common intracellular analytes found that major fluorescence
Scheme 3 Synthesis of probe 6 via the Zincke salt allows the sulfonates
to be introduced in one step after the 4-nitrobenzyl ester trigger group
has been formally installed on the polymethine chain.
enhancement could only be triggered by nitroreductase
enzyme (Fig. 2).§ Three sets of experimental findings showed
that C4′-ester group in 6 is not susceptible to cleavage by ester-
ase enzyme: (1) There was no cleavage of 6 to produce 3 after
incubation with porcine liver esterase (a broad specificity
enzyme) for 24 hours (Fig. S22†). (2) Porcine liver esterase
cleaves the known esterase substrate Calcein AM, whereas 6 is
not an esterase substrate under the same conditions
(Fig. S23†). (3) Probe 6 is not cleaved by the presence of
porcine liver esterase, but a subsequent addition of nitroreduc-
tase to the sample produces a rapid and large increase in NIR
fluorescence due to appearance of cleavage product
3
(Fig. S24†). This latter result suggests that cleavage of probe 6
by intracellular nitroreductase enzyme is substantially more
efficient than any putative probe cleavage by intracellular ester-
ase enzyme.
The capabilities of 5 and 6 as fluorescent NIR probes for
cell hypoxia were tested in a cell culture model. In short, A549
‡The Michaelis–Menten constants for nitroreductase catalysed cleavage of 6
were determined to be Km = 17 µM and Vmax = 0.18 µM s−1. See Fig. S21† for data
and Table S2† for a comparison with related literature NIR fluorescent nitrore-
ductase probes. It was not possible to determine the Michaelis–Menten con-
stants for cleavage of 5 because of extensive concentration-dependent probe self-
aggregation.
Fig. 1 (a and c) Absorption spectra of 5 or 6 (5 μM) in PBS (pH 7.4,
37 °C) supplemented with NADH (500 μM), before and 60 minutes after
addition of nitroreductase (NTR, 1 μg mL−1). (b and d) Fluorescence
spectra of the same samples, along with the change over time of fluor-
§Inspection of Fig. 2 shows that the fluorescence of probe 5 is enhanced slightly
by the presence of rat liver microsomes which is attributed to dequenching of
the lipophilic probe due to interaction with the microsomal membrane. This
effect is not observed with more hydrophilic probe 6.
escence intensity at 780 nm. For 5, λex = 733 nm, and for 6, λex
=
750 nm.
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Fig. 2 Relative fluorescence intensity of (a) 5 or (b) 6 (5 µM) alone (1), or
after addition of Cys (2), Tyr (3), HOCl/OCl– (4), Trp (5), Ser (6), glucose
(7), ascorbic acid (8), Na+ (9), GSH (10), DTT (11), H2O2 (12), NADH + rat
liver microsomes (5 µg mL−1) (13), porcine liver esterase (0.05 µg mL−1
(14), porcine liver esterase (1 µg mL−1) (15), NADH + NTR (0.05 µg mL−1
)
)
(16), NADH + NTR (1 µg mL−1) (17), after 30 minutes in ultrapure water.
For 5 λex = 733 nm. For 6 λex = 750 nm, λem = 782 nm. The concentration
of all added analytes was 5 µM. T = 25 °C. Error bars are too small to be
visualized.
Fig. 3 Representative epifluorescence images of A549 cells that were
treated for 12 hours with either 5 or 6 (5.0 μM) under conditions of nor-
moxia (20% O2) or hypoxia (1% O2). Length scale bar = 20 μm. Red fluor-
escence is the NIR probe and blue fluorescence is cell nucleus stained
with Hoechst dye. The bar graphs show quantification of intracellular
NIR fluorescence intensities as mean pixel intensity (MPI) and include a
comparison with probe-treated hypoxic cell populations that were pre-
treated with the nitroreductase (NTR) inhibitor dicoumarol.
cells (lung adenocarcinoma) were treated with an aliquot of
fluorescent probe, and then maintained under normoxic (20%
oxygen) or hypoxic (1% oxygen) conditions for 12 hours.
Independent protein expression measurements confirmed that
the hypoxic condition raised the amount of intracellular nitror-
eductase by almost three-fold over 12 hours (Fig. S25†).
Fluorescence microscopy experiments using the known and
validated nitroreductase probe 7 (Fig. S29 and S30†) showed
that the hypoxic cells produced about twice the fluorescence
response.25 Separate experiments that treated normoxic and
hypoxic cells with the Cy7 probes 5 and 6 also observed the
same increased level of NIR fluorescence in the hypoxic cells
(Fig. 3). Pretreating the hypoxic cells with the nitroreductase
inhibitor dicoumarol produced no difference in NIR fluo-
rescence compared to normoxic cells (Fig. 3 and S26†), impli-
cating increased intracellular nitroreductase as the reason for
the enhanced fluorescence in hypoxic cells. Cell viability
assays found that Cy7 probe 5 was quite toxic to the cells but
the sulfonated analogue 6 produced no cell toxicity (Fig. 4).
Furthermore, the carboxylic products 1 and 3 were not cell
toxic (Fig. S31†). We suspected that the hydrophobic and cat-
ionic 5 accumulates more in the cell mitochondria and sup-
porting evidence for this hypothesis was gained from separate
Fig. 4 Cell viability assay of A549 cells treated for 12 hours with (a) 5
or (b) 6 at 37 °C, under conditions of hypoxia (1% O2) or normoxia
(20% O2). N = 3.
cell co-incubation studies that treated cells with fluorescent toxicity of probe 5 correlates with increased mitochondria
NIR probe and fluorescent mitotracker green. Fluorescence co- accumulation but presently we have no proof of causality.
localization micrographs indicated substantially higher mito-
To demonstrate the feasibility of 5 and 6 as fluorescent NIR
chondria accumulation in cells treated with probe 5 than cells probes for future in vivo imaging studies we conducted
treated with probe 6 (Fig. S27 and S28†). Thus, the greater cell phantom imaging experiments using a standard animal
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imaging station and found that nitroreductase-activated nitroreductase (NTR from Escherichia coli, purchased from
increases in NIR fluorescence intensity were easily observed as Sigma Aldrich) was prepared in cold ultrapure water and pre-
high contrast images (Fig. S18†). Moreover, constant NIR served at −20 °C. A stock solution of NADH was also prepared
irradiation of separate solutions containing the probe cleavage in cold ultrapure water when needed and stored at −20 °C. An
products 1 or 3, showed comparatively high photostability aliquot of molecular probe (5 µM) was added to a cuvette con-
compared to the clinically approved heptamethine cyanine dye taining 1× PBS Buffer, followed by the addition of NADH
ICG (Fig. S32†), further supporting the feasibility for in vivo (500 µM) and NTR (1 µg mL−1). The final mixture had a total
imaging.
volume of 1 mL and solutions were mixed by inversion for 5
seconds before monitoring changes in absorbance and fluo-
rescence spectra. Enzyme inhibition studies used dicoumarol
(DC) (0.25 mM), a known competitive inhibitor of NTR. The
order of addition was molecular probe (5 µM), DC (0.25 mM),
NADH (500 µM) and NTR (1 µg mL−1).
Conclusions
The combination of good water solubility, low propensity for
self-aggregation, resistance to esterase action, non-toxicity, and
large nitroreductase-triggered enhancement of NIR fluo-
rescence makes sulfonated Cy7 fluorophore 6 a very attractive
choice for further study as a “turn on” fluorescent NIR probe
for imaging hypoxia in living subjects. From a broader per-
spective, the general self-immolation mechanism in Scheme 2
can undoubtedly be expanded beyond 1,6-elimination pro-
cesses to include different types of intramolecular cyclizations
that do not create reactive quinone methide by-products.22,23,26
Collectively, these bond fragmentation processes can be trig-
gered by a wide range of stoichiometric chemical reactions
and enzyme catalyzed cleavage processes,1,7,27 thus enabling
selective NIR fluorescence sensing and imaging of chemical
and enzymatic activity in complex biological media.
Furthermore, it should be possible to substitute the indoli-
nene units at each end of the Cy7 fluorophore with additional
π-extended units that extend the absorption/emission wave-
lengths beyond 1000 nm and thus create bioresponsive probes
for the NIR II region.6 Typically, these π-extended units will
increase molecular hydrophobicity but this potential drawback
can be countered by the presence of sulfonate groups as exem-
plified by structure 6. In this regard, it is worth emphasizing
the major synthetic advantage gained by preparing sulfonated
bioresponsive Cy7 probes by the strategy employed in this
study; i.e., using Zincke salt methodology to introduce the sul-
fonates in one step after the trigger group (Scheme 3).5 This
synthetic innovation provides convenient access to the desired
water-soluble bioresponsive NIR probes with the non-toxicity
and favorable physiochemical and pharmacokinetic properties
that are necessary for eventual clinical translation. Studies that
explore these opportunities are underway and the results will
be reported in due course.
Cell microscopy studies
Cell culture conditions. A549 (ATCC® CCL-185™) human
lung adenocarcinoma cells were obtained and cultured and
maintained in F12K Media supplemented with 10% fetal
bovine serum, 1% penicillin streptomycin. Culture was done at
37 °C, air supplemented with 5% CO2.
Sample preparation and microscopic imaging. A549 cells
were plated onto an 8-well chambered coverglass (Lab-Tek,
Nunc) and grown to 80% confluency (48 hours). The medium
was then removed, and the cells were washed with PBS twice.
Separate cell samples were incubated with 5 µM of molecular
probe in warm F12K media at 37 °C for 12 hours in a chamber
that maintained hypoxic (1% O2) or normoxic (20% O2) atmo-
sphere (also containing 5% CO2). After incubating the cells
with probes, the media was removed, and cells were washed
three times with PBS. Cells were then stained with Hoechst dye
(3 µM) for 10 min, and finally washed three times with PBS
and imaged under Opti-mem Media using a Zeiss Axiovert 100
TV epifluorescence microscope (63×) equipped with a DAPI
filter (ex: 445/40, em: 494/20) to image the nucleus and ICG
filter (ex: 769/41, em: 832/37) to image NIR fluorescence. All
experiments were repeated three times and micrographs of 6
separate viewing fields were obtained during each trial and
analyzed using image J software. For each micrograph, a back-
ground subtraction with a rolling ball radius of 20 pixels was
applied. Next, a triangle threshold was employed and used to
calculate the average mean pixel intensity (MPI) where overall
averages and SEM were calculated and plotted in GraphPad
Prism. p values were calculated using t tests with ANOVA.
Intracellular nitroreductase inhibition studies. A549 cells
were plated onto an 8-well chambered coverglass and grown to
80% confluency (48 hours). The medium was then removed,
and the cells were washed with PBS twice. Separate cell
samples were incubated with 0.1 mM dicoumarol (DC) for
30 minutes in media. The media was then removed, and cells
were washed three times with PBS. Cells were treated with an
aliquot of 5 or 6 (5 µM) in warm F12K media and then incu-
bated at 37 °C for 12 hours under hypoxic atmosphere (1%
O2). After incubating cells with probes, the media was
removed, and cells were washed three times with PBS. Cells
Experimental
Synthesis and compound characterization
All synthetic procedures and compound characterization are
provided in the ESI.†
Enzyme cuvette studies
Separate stock solutions of molecular probes 5, 6, and 7 were were then stained with Hoechst dye (3 µM) for 10 min, and
prepared in dimethyl sulfoxide (DMSO). A stock solution of finally washed three times with PBS and imaged under Opti-
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mem Media. The ICG filter set was used to image the NIR fluo-
rescence and DAPI filter was used to image the nucleus. All
Acknowledgements
experiments were repeated three times and images of 6 separ- We are grateful for funding support from the US NIH
ate viewing fields were obtained during each trial.
(R35GM136212 and T32GM075762).
Colocalization assay of cells. A549 cells were plated onto an
8-well chambered coverglass and grown to 80% confluency
(48 hours). The media was then removed, and the cells were
washed with PBS twice. Cells were treated with an aliquot of 5,
or 6 (5 µM) in warm F12K media and then incubated at 37 °C
for 12 hours under hypoxic (1% O2) atmosphere. Then, the
media was removed and cells were washed three times with
PBS. Mitotracker Green (100 nm) was added to each well and
cells were incubated at 37 °C for 15 minutes under hypoxic
(1% O2) atmosphere. Cells were then washed three times with
PBS and subsequently stained with Hoechst dye (3 µM) for
10 min, and finally washed three times with PBS and imaged
under Opti-mem Media. The ICG filter set was used to image
the NIR fluorescence of 5 or 6 and FITC filter set was used to
image the Mitotracker Green. Pearson’s correlation coefficients
and Mander’s coefficients (M1 and M2) were calculated using
the JACoP plugin in image J (Fig. S27 and S28†). M1 is the
ratio of the total intensity of pixels from 5 or 6 which overlaps
with the total intensity of the mitotracker. M2 is the opposite
which shows total overlap of the mitotracker with 5 or 6. All
experiments were repeated three times and images of 6 separ-
ate viewing fields were obtained during each trial.
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Conflicts of interest
There are no conflicts to declare.
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