Thiophene bridged hydrocyanine-a new fluorogenic ROS probe

Article abstract of DOI:10.1039/c7cc04847e

Hydrocyanines are a class of commonly used reactive oxygen species (ROS) fluorescent imaging probes, which can image ROS in cell culture, organ culture, and in vivo. However, despite their widespread use, hydrocyanines have several drawbacks that limit their effectiveness, such as a high rate of auto-oxidation, a small Stokes shift, and poor water solubility. In addition, the hydrocyanines oxidize into cyanine dyes, which themselves decompose in the presence of ROS, and this further lowers their sensitivity towards detecting ROS. In this report, we present a new hydrocyanine analog, termed as thiophene-bridged hydrocyanine (TBHC), which has its double bonds replaced with a bisthiophene. TBHC is 8.06-fold more stable to auto-oxidation than the hydrocyanine hydro-Cy5 and is significantly better at imaging ROS in cell culture.

Full text of DOI:10.1039/c7cc04847e

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Thiophene bridged hydrocyanine – a new
fluorogenic ROS probe†
Cite this:DOI: 10.1039/c7cc04847e
a
a
a
Santanu Maity,
Subhamoy Das,
Corinne M. Sadlowski,
Jingtuo Zhang,a
Giri K. Vegesnab and Niren Murthy*a
Received 22nd June 2017,
Accepted 21st August 2017
DOI: 10.1039/c7cc04847e
rsc.li/chemcomm
Hydrocyanines are a class of commonly used reactive oxygen imaging community. For example, hydrocyanines are synthesized
species (ROS) fluorescent imaging probes, which can image ROS in one step from cyanines, and have tunable emission wave-
in cell culture, organ culture, and in vivo. However, despite their lengths. The hydrocyanines were able to image ROS in a variety
widespread use, hydrocyanines have several drawbacks that limit of animal models, in cell culture, and in organ culture, and were
their effectiveness, such as a high rate of auto-oxidation, a small used to elucidate the role of ROS in numerous cell-signaling
Stokes shift, and poor water solubility. In addition, the hydrocyanines pathways.31–34 However, despite their widespread use, hydro-
oxidize into cyanine dyes, which themselves decompose in the cyanines have several drawbacks that limit their efficacy: (1) they
presence of ROS, and this further lowers their sensitivity towards have a high rate of auto-oxidation, which causes high levels of
detecting ROS. In this report, we present a new hydrocyanine analog, background fluorescence,30,39 (2) they oxidize into cyanine dyes
termed as thiophene-bridged hydrocyanine (TBHC), which has its that decompose in the presence of ROS,35–38 (3) the cyanine dyes
double bonds replaced with a bisthiophene. TBHC is 8.06-fold more have a small Stokes shift, which increases their background
stable to auto-oxidation than the hydrocyanine hydro-Cy5 and is fluorescence, and (4) membrane permeable hydrocyanines have
significantly better at imaging ROS in cell culture.
poor water solubility,35,39 which makes them challenging to use
with cells and in vivo. Therefore, there is a great need for
Radical oxidants are implicated in over 150 diseases and are also developing a second generation of hydrocyanine probes, which
involved in fundamental physiological processes such as inflam- are stable to auto-oxidation, have low background fluorescence,
mation, aging, apoptosis, cell growth, and cell differentiation.1–17 and generate oxidation products that are stable in the presence
There is therefore great interest in imaging the concentration of of ROS. In this report, we present a new hydrocyanine analog,
radical oxidants in cells.18–23 Several fluorescent dyes have been termed as thiophene-bridged hydrocyanine (TBHC), which is
developed to image radical oxidants, which are based on the stable to auto-oxidation, has a large Stokes shift and also has the
amine oxidation reaction. The amine oxidation reaction converts appropriate hydrophobic/hydrophilic balance needed for imaging
a carbon–nitrogen single bond into a carbon–nitrogen double ROS in cell culture and in vivo.
bond via a process initiated by the one-electron oxidation of the
We hypothesized that the polymethine linker connecting
nitrogen. The amine oxidation reaction is a powerful method for the two substituted indole rings of the hydrocyanines was the
developing dyes that detect radical oxidants and the most commonly potential cause of their auto-oxidation and the instability of
used radical oxidant imaging probes, such as dihydroethidium their oxidized products. A new hydrocyanine derivative was
(DHE), hydrocyanines and reduced cyanine-hybrid dyes all detect therefore synthesized, termed as thiophene bridged cyanine
radical oxidants via the amine oxidation reaction.18,23–25
(TBC), which had its polymethine linker replaced with a
The hydrocyanines are a class of radical oxidant probes bisthiophene containing an electron-donating carboxymethyl
based on the amine oxidation reaction, which can detect radical phenyl ether group (Fig. 1). TBC is a new fluorescent dye that
oxidants in cell culture, in organ culture, and in vivo and have causes intramolecular charge transfer (ICT) between the carboxy-
found widespread applications in the biological community.26–30 methyl phenyl ether group and the iminium cation and needs
Hydrocyanines have several beneficial properties that have enabled the electron-withdrawing strength of the iminium cation to
their rapid commercialization and use throughout the ROS generate intramolecular charge transfer. Reduction of TBC’s
iminium cation with sodium borohydride generates a non-
fluorescent compound (termed TBHC), which shows weak
fluorescence because its iminium cation has been converted
a University of California, Berkeley, USA. E-mail: nmurthy@berkeley.edu
b Profusa, Inc., 345 Allerton Avenue, South San Francisco, CA 94080, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc04847e into a tertiary amine. A tertiary amine is an electron donor,
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Fig. 2 Synthesis of TBHC (1) and TBC (2). (a) Pd(PPh3)4, CsF, DMF, 80 1C,
82%; (b) aniline, toluene, reflux, quantitative; (c) 2,2,3-trimethyl-N-hexyl-
indeloic acid, acetic anhydride, 80 1C, 20%; (d) porcine liver esterase
(1000 unit), PBS 82%; (e) NaBH4, methanol, 63%.
Fig. 1 Thiophene bridged hydrocyanine (TBHC): a new hydrocyanine
analog that images reactive oxygen species with superior performance.
The hydrocyanines are fluorescent probes that image ROS. However, their
auto-oxidation, poor water solubility, and small Stokes shift limits their
efficacy. In this report, we present a new hydrocyanine analog, termed as
the thiophene bridged hydrocyanine (TBHC), which has the polymethine
bridge of a hydrocyanine replaced with a bisthiophene. TBHC is stable to
auto-oxidation, has a large Stokes shift, and the appropriate hydrophobic/
hydrophilic balance needed for imaging ROS in cell culture and in vivo.
reacted with aniline in toluene to obtain the intermediate
N-phenylimine 6, which was subsequently fused with N-hexa-
2,3,3-trimethyl indoleic acid 7 in acetic anhydride to yield the
methyl ester of TBC 8. Compound 8 was quantitatively converted
to TBC by enzymatic hydrolysis. TBHC was synthesized by a
regioselective reduction of the iminium cation of TBC in
methanolic sodium borohydride (see Scheme S1, ESI†).
The absorption and fluorescence spectra of TBHC and its
ROS-mediated oxidation product TBC were investigated. Fig. 3a
demonstrates that TBC has a fluorescence emission maximum
at 700 nm and an absorption maximum at 573 nm (see Fig. S9,
ESI†), whereas TBHC showed a weak fluorescence emission at
700 nm, which was 28-fold lower than that of TBC. The
difference in the fluorescence quantum yield between TBC
and TBHC at 700 nm is expected based on the differences in
the electron-withdrawing ability between the iminium cation of
TBC and the tertiary amine of TBHC. We measured the quantum
yield and Stokes shift of TBC in PBS and 10% (w/v) albumin
as opposed to an electron withdrawing group, and TBHC therefore
cannot cause intramolecular charge transfer and consequently
shows weak fluorescence. However, in the presence of ROS, TBHC
undergoes an amine oxidation reaction and regenerates its
iminium cation, which causes a dramatic increase in the
fluorescence quantum yield. A thiophene was selected as a
replacement for the polymethine linker of the hydrocyanines
because thiophenes have excellent photostability and chemical
stability and should therefore be stable to ROS. In addition,
thiophenes that cause intramolecular charge transfer have a
large Stokes shift and can be easily synthesized with a variety of
substituent groups to generate probes with the appropriate
hydrophobic/hydrophilic balance for cell and animal imaging.
TBHC should have significant advantages over traditional
hydrocyanines because it contains a chemically inert bisthiophene
instead of a polymethine chain. TBHC and its oxidized product
TBC should therefore have minimal problems with auto-oxidation
and decomposition, and should also have a large Stokes shift. In
addition, TBC has a log P of 1.31, which is an ideal hydrophobicity
for intracellular ROS imaging applications. TBHC is hydrophobic
enough to be membrane permeable, yet hydrophilic enough to be
dissolved in water. In contrast, traditional hydrocyanines are either
so hydrophobic that they require toxic organic solvents for delivery
or have sulfonic acid groups, which are permanently charged and
membrane impermeable.35,39
The synthesis of TBHC (Fig. 2) was accomplished in five
steps with an overall yield of 11%. A Suzuki coupling between
boronic ester 4 and the iodinated bisthiophene 3 was performed
Fig. 3 TBHC can detect the hydroxyl radical with nanomolar sensitivity.
(a) Fluorescence spectra of 50 mM of TBC and TBHC respectively in PBS,
TBC has a 28-fold higher fluorescence at 700 nm, excitation at 573 nm.
to yield the bisthiophene aldehyde 5. Compound 5 was then (b) TBHC can detect the hydroxyl radical with nanomolar sensitivity (35 nM).
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(in PBS). 10% (w/v) albumin was selected as a solvent to
measure the quantum yield of TBC because the cellular
environment contains a high protein concentration, which will
affect the quantum yield of TBC. TBC showed a quantum yield
of 2.3% in 10% (w/v) albumin (0.11% in PBS), whereas TBHC
showed a quantum yield of only 0.005% in 10% albumin. In
addition, TBC has a 127 nm Stokes shift, which is significantly
larger than that of Cy5 and should, therefore, generates lower
levels of auto-fluorescence in cells than Cy5.
We investigated the ability of TBHC to detect the hydroxyl
radical. TBHC (300 mM) was mixed with various concentrations
of hydrogen peroxide (1–100 nM), and 4 units of horseradish
peroxidase and the resulting increase in fluorescence was
measured at 700 nm after 15 min of incubation (see Fig. S10,
ESI†). Fig. 3b demonstrates that TBHC can detect the hydroxyl
radical with a detection limit of 35 nM, which is comparable to
Fig. 5 TBC is stable to ROS-mediated decomposition, and TBHC quantitatively
regenerates TBC in the presence of ROS. (a) A 25 mM solution of TBC and Cy5
was treated with 250 mM of ꢀOH, ÀONOO and ÀOCl and their decomposition
was measured. TBC is protected from ROS-mediated decomposition. (b) A 25 mM
of TBHC and hydro-Cy5 were treated with 10 equivalents of the hydroxyl radical
and the regeneration of TBC and Cy5 was determined. TBHC efficiently
regenerates TBC, and generated TBC with 75% efficiency, whereas hydro-Cy5
generated Cy5 with only a 17% efficiency.
the detection sensitivity of the hydrocyanines. Therefore, TBHC in cells experiencing oxidative stress due to the production of
À
has the properties needed to measure the cellular levels of ROS.
ONOO, OH, or ÀOCl. The decomposition of cyanine dyes in
ꢀ
In addition, we determined the ROS specificity of TBHC. TBHC cells experiencing oxidative stress will limit the sensitivity of the
was incubated with a variety of ROS species and the resulting hydrocyanines towards detecting ROS. We, therefore, investigated
increase in fluorescence was measured. Fig. S11 and S13 (ESI†) whether TBC was more stable to ROS than a cyanine dye. TBC and
demonstrate that TBHC is specific to hydroxyl/superoxide Cy5 were incubated in the presence of ÀONOO, ꢀOH, and ÀOCl
radical oxidants, similar to the hydrocyanines, which suggests for 45 minutes and the decomposition of TBC and Cy5 was
that TBHC oxidizes via an amine oxidation based mechanism, determined by measuring their change in absorption. Fig. 5a
similar to the hydrocyanines.
demonstrates that Cy5 was 75% degraded in the presence of the
The auto-oxidation of the hydrocyanines is a key challenge hydroxyl radical, whereas TBC was only 15% degraded under the
that limits their effectiveness at imaging ROS in cell culture. We same conditions. Thus, TBC is significantly more stable to ROS
therefore measured the auto-oxidation of TBHC in PBS and than Cy5. The bisthiophene bridge of TBC, therefore, stabilizes
compared it against hydro-Cy5. An 80 mM solution of TBHC or it from ROS-mediated decomposition.
hydro-Cy5 was incubated at RT for 48 hours and their respective
We investigated if the stability of TBC towards ROS-mediated
increase in absorbance was measured. Fig. 4 demonstrates that decomposition allowed TBHC to quantitatively regenerate TBC
the auto-oxidation half-life of TBHC was 250 hours, whereas in the presence of ROS, and compared its fluorescence recovery
hydro-Cy5 had an auto-oxidation half-life of only 31 hours to that of hydro-Cy5. The percentage of fluorescence recovery of
ꢀ
(Fig. S12, ESI†). TBHC is 8.06-fold more stable to auto-oxidation TBHC and hydro-Cy5 in the presence of OH was investigated.
than hydro-Cy5, and replacing the polymethine linker of hydro-Cy5 TBHC or hydro-Cy5 (25 mM) were mixed with 300 mM H2O2 in
with a bisthiophene therefore significantly increases its stability to the presence of 8 units of HRP, and the resulting fluorescence
auto-oxidation.
was compared with that of a 25 mM solution of either TBC or
Another potential limitation of the hydrocyanines is that hydro-Cy5. Fig. 5b demonstrates that 72% of TBHC was oxidized
they oxidize into cyanine dyes, which themselves are unstable to TBC in the presence of ROS. In contrast, only 17% of hydro-
in the presence of ROS. For example, Nagano et al.38 and several Cy5 was oxidized into Cy5 under the same conditions. TBHC
other groups have demonstrated that cyanine dyes decompose therefore efficiently generates TBC in the presence of ROS and
has great potential for quantitative imaging of ROS.
We evaluated the toxicity of TBHC (see Fig. S13, ESI†) and
its ability to image ROS in macrophages stimulated with lipo-
polysaccharide (LPS) and compared its efficacy against hydro-Cy5.
Raw 264.7 macrophages were treated with LPS for 16 hours, and
subsequently treated with a 100 mM of TBHC or hydro-Cy5 and
imaged. The controls for this experiment were cells treated with
phosphate buffer saline (PBS) and TBHC and cells treated with LPS +
TEMPOL and TBHC. Fig. 6 and Fig. S15, S16 (ESI†) demonstrate
that TBHC is better than hydro-Cy5 at imaging ROS in cell culture.
For example, TBHC generated a 100% increase in fluorescence
intensity between the cells treated with LPS versus control cells,
whereas hydro-Cy5 only generated a 35% increase. In addition,
Fig. 4 TBHC is stable to auto-oxidation. An 80 mM solution of TBHC or
hydro-Cy5 was incubated in PBS and their absorbances were measured
over 48 hours. 89% of TBHC was intact after 48 hours, whereas only 42%
the ROS scavenger TEMPOL was able to inhibit the increase
in fluorescence generated from LPS in the TBHC treated cells,
of hydro-Cy5 remained intact. TBHC is 8.06-fold more stable in PBS to
auto-oxidation than hydro-Cy5.
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Fig. 6 TBHC can image ROS produced from lipopolysaccharide (LPS) stimulated macrophages (figure width 50 mm). (a–c) RAW 264.7 macrophages
were treated with PBS or LPS + TEMPOL or LPS and imaged with TBHC (100 mM). Cells treated with LPS and TBHC have a high level of fluorescence.
(d and e) RAW 264.7 macrophages were treated with LPS or PBS and imaged with hydro-Cy5 (100 mM). (f) Cells treated with TBHC + LPS had a 100%
increase in relative fluorescence over PBS or LPS + TEMPOL treated cells. (g) Cells treated with TBHC + LPS had a 35% increase in relative fluorescence
over PBS treated cells.
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In summary, we present a new hydrocyanine analog, termed
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