Development of a water-soluble 3-formylBODIPY dye for fluorogenic sensing and cell imaging of sulfur dioxide derivatives

Article abstract of DOI:10.1016/j.tetlet.2019.04.039

A new water-soluble, highly fluorogenic 3-formylBODIPY dye that enables the sensing of SO₂ derivatives in aqueous buffers and cancer cells is reported. The quaternary ammonium group appended through the meso-position of the BODIPY dye ensures water solubility. The probe exhibits high specificity for cytosolic (bi)sulfites and fluoresces brightly in human lung adenocarcinoma cells (A549).

Full text of DOI:10.1016/j.tetlet.2019.04.039

Accepted Manuscript
Development of a water-soluble 3-formylBODIPY dye for fluorogenic sensing
and cell imaging of sulfur dioxide derivatives
Murat Iş ık, Ilke Simsek Turan, Suay Dartar
PII:
S0040-4039(19)30387-9
https://doi.org/10.1016/j.tetlet.2019.04.039
TETL 50754
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 February 2019
15 April 2019
24 April 2019
Please cite this article as: Iş ık, M., Simsek Turan, I., Dartar, S., Development of a water-soluble 3-formylBODIPY
dye for fluorogenic sensing and cell imaging of sulfur dioxide derivatives, Tetrahedron Letters (2019), doi: https://
doi.org/10.1016/j.tetlet.2019.04.039
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Development
of
a
water-soluble
3-
Leave this area blank for abstract info.
formylBODIPY dye for fluorogenic sensing and
cell imaging of sulfur dioxide derivatives
Murat Işıka, *, Ilke Simsek Turanb and Suay Dartarc

1
Tetrahedron Letters
Development of a water-soluble 3-formylBODIPY dye for fluorogenic sensing and
cell imaging of sulfur dioxide derivatives
Murat Işıka, , Ilke Simsek Turanb and Suay Dartarc
aDepartment of Food Engineering, Bingöl University, Bingöl, 12000, Turkey
bUNAM-National Nanotechnology Research Center, Bilkent University, Ankara 06800, Turkey
cDepartment of Chemistry, Faculty of Science, İzmir Institute of Technology, Urla, 35430 İzmir, Turkey
This work is dedicated to the memory of our beloved mentor, Prof. Dr. İdris Mecidoğlu Akhmedov, who recently passed away.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A new water-soluble, highly fluorogenic 3-formylBODIPY dye that enables the sensing of SO2
derivatives in aqueous buffers and cancer cells is reported. The quaternary ammonium group
appended through the meso-position of the BODIPY dye ensures water solubility. The probe
exhibits high specificity for cytosolic (bi)sulfites and fluoresces brightly in human lung
adenocarcinoma cells (A549).
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
fluorogenic probes
BODIPY dyes
water-soluble fluorophores
sulfur dioxide derivatives
The fluorogenic sensing and signalling of biologically relevant
small molecules, with a major emphasis on sulfur-containing
ones, has drawn significant attention.1 Being both a well-known
air pollutant and a common food additive (E220–228), sulfur
dioxide (SO2), has been the focus of many recent works.2 Once
foods/beverages or in biological matrices is of considerable
interest.
Early methods for the detection of sulfites include titration
(first oxidation by hydrogen peroxide to sulfuric acid, then
titration with sodium hydroxide),8 electrochemistry,9 capillary
electrophoresis,10 spectrofluorometry,11 and colorimetry.12
However, these methods generally require pretreatment of the
samples, i.e. extraction or bleaching, which often retards the
analysis to hours.
hydrated, it is transformed into a pH-dependent equilibrium
2–
mixture of sulfite, bisulfite and/or sulfur dioxide species (SO3
,
HSO3– and/or SO2), all of which are collectively called sulfites or
sulfiting agents, and have found extensive use as preservatives in
food processing for more than a century.3 The wine industry, for
example, relies heavily on sulfites to improve/maintain the
sensory characteristics of end-products, such as taste, aroma, and
color.4 Exposure to high doses of sulfites, on the other hand, is
associated with a number of diseases such as allergic asthma,
urticarial dermatitis, hypotension, abdominal pain, lung cancer,
and neurological disorders.5 At some extremes, it is even known
to kill individuals who are hypersensitive to sulfites.5
Consequently, the use of sulfites as antimicrobial agents/food
additives is restricted by international legislation (upper limit =
0.7 mgkg-1 of body weight; World Health Organisation—WHO).3
Additionally, sulfur dioxide is known to be endogenously
produced in living organisms via the metabolic degradation of
biological thiols (e.g. cysteine and glutathione).6 As such, recent
reports have revealed vasodilatation activities of SO2, and
thereby suggest its role as a gasotransmitter.7 Nevertheless, its
precise physiological roles remain largely unknown.2 Considered
Fluorogenic sensing methods, present excellent opportunities
due to their unique attributes, such as operational simplicity,
inherent high sensitivity, and ready availability of fluorescence
spectrometers; more importantly, fluorogenic probes make in
vivo imaging possible.13 The design of fluorescent probes relies
largely on reaction-based sensing platforms,14 which take
advantage of the nucleophilicity of the sulfur atom of sulfiting
agents. Several sulfite-reactive functional groups (Michael type
acceptors,15 levulinate esters,16 aldehydes,17 and others18) have
been installed on fluorophores to create a sensing event, among
which, protocols involving conjugate addition are by far the most
frequent. Early examples of sulfite probes, however, have
required the use of an organic co-solvent,16b,17c long incubation
times (up to 1 day),18d UV excitation,18a and exhibited low
fluorescence enhancement often with nonspecific side reactions,
which limit their practical applications. Therefore, to avoid the
above-listed weaknesses of these precedents, the last couple of
t—og—ethe—r, the sensing and quantification of sulfites in samples of
 Corresponding author. Tel.: +90-426-216-1962; fax: +90-426-216-0033; e-mail: misik@bingol.edu.tr

2
years have witnessed a number of papers on the fluorogenic
sensing of sulfites.15–18
Scheme 1. Synthesis of probes 1–4.
using a method developed by Ziessel and co-workers.29 We
discovered that the use of twelve equivalents of DDQ was
essential to obtain compound 8 in acceptable purity for the next
step. Finally, the benzylic tert-amino groups of the 2- and 3-
formylBODIPY dyes (7 and 8) were quaternized by treating their
concentrated dichloromethane solutions with 10 equiv. of
(pseudo)-halides (methyl iodide and 1,3-propanesultone) at room
temperature for 2 days. This series of reactions furnished the
targeted probe candidates 1–4, the purification of which was
achieved by repetitive centrifuging-washing processes.
Figure 1. Design of the sulfite probes.
Building on our previous efforts in this area,19 herein we wish
to describe new water-soluble 2-/3-formyl-BODIPY dyes (Fig.
1). BODIPY (boron-dipyrromethene) dyes were explored
because of their highly desirable characteristics, such as narrow
emission bands, rich functionalization chemistry, high molar
absorptivity and quantum yields, no pH dependency and
operation in the visible spectral range.20 These dyes, once
judiciously designed, allow control over the fluorescence
emission intensity through photoinduced electron transfer (PeT)
and/or internal charge transfer (ICT) photophysical processes.21
BODIPY dyes find use in a number of applications such as
fluorescent probes/labels,22 dye sensitized organic solar cells,23
and photodynamic therapy,24 among many others.25
All of the (zwitter)-ionic compounds 1–4 were soluble in
water, thus permitting all spectroscopic studies to be conducted
in aqueous buffers. We began our spectroscopic measurements
by taking the fluorescence emission spectra of free dyes 1–4 in
phosphate buffered saline (PBS; 10 mM) at physiological pH
(7.4) (see ESI for UV-vis and fluorescence excitation and
emission spectra of compounds 1–4). Figure 2 shows the relative
emission behaviour of compounds 1–4, where each is present at
equal concentration (1 μM). 3-FormylBODIPY 3, among the
candidates, lacks background fluorescence and thus was selected
as the probe of choice. The others, however, showed significant
fluorescence emissions visible to the naked eye (see inset in Fig.
2). Probe 3, once incubated with 1000 equiv. of sulfite for two
minutes, starts to fluoresce greenish-yellow at 543 nm (see ESI).
The equilibrium distribution of sulfite species strongly depends
on the pH of the medium (vide supra), and bisulfite addition to
probe 3 (aldehyde) may also show similar pH-dependency since
the reaction is reversible (Eq. 1 in Fig. 1). Therefore, we wanted
to explore the pH dependency of this sensing event. To this end,
we screened a pH range of 4–8 by altering the pH in half unit
increments (see ESI), and gratifyingly the probe was essentially
insensitive to variations in pH in the absence and presence of
Their insolubility in water, however, impedes potential
applications in aqueous media. They typically tend to form non-
fluorescent aggregates, unless a water-solubilizing auxiliary
group is attached.19,26 Encouraged by parallel efforts of the
Ziessel group,26f we have introduced water-solubilizing (zwitter)-
ionic quaternary ammonium units through the meso- (or 8-)
position of the BODIPY core to make them water-soluble. The
reactivity with sulfite was ensured by conjugating the
fluorophore with formyl units through the 2- and 3-positions of
the BODIPY core. We hypothesized that, upon reaction with
sulfites, the π-conjugation between the BODIPY core and the
formyl unit would be broken, which resultantly may by-pass an
ICT quenching process and induce a change in fluorescence
emissions.
Therefore,
we
synthesized
4-
((dimethylamino)methyl)benzaldehyde,
a
common starting
material for all probe candidates 1–4, using a three-step patent
procedure.27 In addition, we tried a one-step synthesis of this
aldehyde by direct reductive amination of terephthalaldehyde; the
strategy worked but with low chemical yields (8–11%, see ESI).
Classical BODIPY dye synthesis conditions, for which catalytic
amounts of trifluoroacetic acid (TFA) is generally sufficient,
failed to give intermediate products 5 and 6 in our hands
(Scheme 1). We reasoned that the benzylic amino group of the
dye buffers the TFA used, thus inhibiting the condensation
reaction of the aldehyde with the pyrroles. As a result, one
equivalent of TFA was necessary to afford BODIPYs 5 and 6 in
reasonable yields (40% and 48% respectively). The available 2-
position of compound 5 was formylated using the Vilsmeier-
Haack formylation protocol reported by Jiao and Hao.28
–
HSO3 /SO32– species. Although the sensing reactions equilibrated
more rapidly at pH ≥6.0 (response time <1 min.) than those at pH
≤5.5 (response time <5 min.) with no significant difference in the
emission profiles, the pH value was set to 4.5 as it provided
higher and more stable emission signals with acceptable response
times (≈5 min.). Given the strong pH-dependency of some
precedent probes, the use of probe 3 would be highly
advantageous for applications where pH independency is
required (e.g. some cancer cells are acidic, while some are
alkaline). The sensing reaction between probe 3 and bisulfites is
amenable to HRMS analysis (see ESI), attesting to the 1,2-
addition.
To reveal the time-dependency of probe 3, we recorded the
–
kinetic profiles of the probe against varying amounts of HSO3
The formyl functionality was introduced to the 3-position of
BODIPY 6 via DDQ-mediated oxidation of an α-methyl group,
(0, 100, 500 and 1000 equiv.). Figure 2 clearly shows that the

3
sensing reaction equilibrates within five minutes when the dye is
at a 5 μM concentration.
Fe3+, and Hg2+), whether by reactions or noncovalent
interactions (see ESI).
We were also interested in the fluorescence response of probe
3 to increasing bisulfite concentrations. Remarkably, probe 3
responds to increasing bisulfite concentrations in a linear fashion
(10–2000 μM), which may suggest the quantification of sulfites
in food/beverage or biological matrices (see Fig. 3). The limit of
detection of probe 3 for sensing bisulfite was determined as 9.05
μM (see ESI). Literature examples of 2- and 3-formylBODIPY
dyes are rare, and they are generally converted to their more
reactive derivatives for sensing applications.30 There only two
reports which make use of 2- and 3-formylBODIPY dyes as
fluorogenic probes.31,32 The groups of Hao and Jiao have shown
that 3-formylBODIPY dyes are able to sense reactive biological
thiols, cysteine and homocysteine [(L)-Cys and (L)-Hcy],
however water-insolubility of the probes requires the use of as
much as 50% methanol as the solvent [HEPES/MeOH, 1/1 (v/v)],
which results in significant initial fluorescence and slow response
(60 min.) towards Cys and Hcy with 3–5 fold fluorescence
intensity enhancement.31 When compared to the 3-
formylBODIPY dyes of Jiao and Hao, probe 3 exhibits enhanced
water-solubility, allows sensing applications in 100% aqueous
media and is quenched very well, which renders it highly
fluorogenic (up to 38 fold enhancement).
800
700
600
500
400
300
200
100
0
800
700
600
500
400
300
200
100
at pH 4.5, 3 only
1
2
3
4
at pH 4.5, 3 + 25 eq bisulfite
at pH 4.5, 3 + 50 eq bisulfite
at pH 4.5, 3 + 100 eq bisulfite
1
2 3 4
0
100 200 300 400 500
500
600
700
800
Wavelength (nm)
Time (s)
Figure 2. Left: Fluorescence emission spectra of the probe
candidates 1–4 (each 1 μM) in 10 mM PBS (pH = 7.40, λex 490 nm).
Slit width: dex = 5 nm, dem = 2.5 nm Inset: Digital photograph of the
solutions under a handheld UV lamp (@ 366 nm). Right: Time-
dependent fluorescence emission spectra of the probe 3 (5 μM) in the
absence and presence of varying amounts of bisulfite in 10 mM
acetate buffers (pH = 4.5, λex 490 nm, λem 543 nm).
Finally, we examined whether probe 3 would be suitable for
applications in live cells. To this end, human lung
adenocarcinoma (A549) cells were incubated at 37 °C first with
probe 3 (5.0 μM) for 30 min, then with bisulfite (500 μM) for
another 30 min. Fluorescence microscopy images (Fig. 4) clearly
indicates that probe 3 is cell-permeable, and, when incubated
alone with cells (panel a), shows no emission suggesting no non-
specific binding, and is able to highlight the cytoplasm of A549
cells in the presence of bisulfites. The probe is nucleus-
impermeable as evidenced by nucleus-staining control
experiment (panel b–d).
We also wanted to see to what extent probe 3 was selective.
Towards this end, a large number of anions, cations, and neutral
molecules with potentially interfering or competing natures were
tested. The spectral responses of the highly competitive anions
and biological thiols are plotted in Figure 3 (Left).
800
Fluorescence emission intensity at 543 nm
180
2000 M HSO3
700
160
a.HSO5 b.H2O2 c.S2O3 d.HSO4 e.NO2 f.CNg.F
h.N3 i.Glucose j.(L)-Lys k.(L)-Cys l.(L)-Hcy m.GSH
600
140
n.HS o.HSO3
120
100
80
60
40
20
0
500
400
300
200
100
0
10 M HSO3
500
550
600
650
3 onlya
b
c
d
e
f
g
h
i
j
k
l m n o
Wavelength (nm)
Anionic/neutral competitors
Figure 3. Left: Fluorescence emission response of probe 3 (2 μM) in
the absence and presence of 100 equiv. of bisulfite and other
potentially competing anions and neutral molecules. GSH:
Glutathione, (L)-Hcy: (L)-Homocysteine, (L)-Cys: (L)-Cysteine
Right: Fluorescence emission response of probe 3 (5 μM) titrated
against increasing amounts of bisulfite (50–2000 μM). Inset: Digital
photograph of the free dye 3 (left) and dye 3 plus an excess of
bisulfite (right) under a handheld UV lamp (@ 366 nm).
Figure 4. Fluorescence images of human lung adenocarcinoma cells
(A549). Fluorescence image of a) A549 cells treated with probe 3
only (5µ M), b) cells treated with DNA-binding 4′,6-diamidino-2-
phenylindole (DAPI) dye (control), c) cells treated with probe 3 (5
µM) and HSO3– (500 µM); d) merged images of frames b) and c).
(λex = 480 nm)
Probe 3 (2 μM) shows a clear preference for bisulfite among
the twenty competing species (100 equiv.) examined; a small yet
detectable response to HS– is also observed. Notably, the probe
–
tolerates reactive oxygen species (ROS: HSO5 , H2O2) and
–
reactive sulfur species (RSS: S2O32–, HSO4 , HS–, (L)-Cys, (L)-
In conclusion, a novel method is reported for the synthesis of
BODIPY dyes that are both water-soluble and effectively
quenched (fluorescence). Four formyl-attached BODIPY dyes 1–
4 bearing quaternary ammonium units which make them water-
soluble were synthesised. 2-Formyl dyes (1 and 2) were highly
–
Hcy, GSH) as well as nucleophiles (CN–, NO2 ) with no
observable signal change. It also displays no spectral interference
to six cations of biological significance (Mg2+, Ca2+, Zn2+, Cu2+,

4
Hou, J.-T.; Li, L.-L.; Lu, C.-Y.; Xie, Y.-M.; Wang, X.; Yu, X.-
fluorescent rendering themselves unsuitable for sensing
applications. Upon reaction with sulfites, otherwise non-
fluorescent probe 3 begins to fluoresce (up to 38 fold) greenish-
yellow (λem = 543nm) within a few minutes. Probe 3 shows high
specificity for sulfites over a number of competitors with no pH-
dependency and works inside cytosol of A549 carcinoma cells,
which makes it an interesting probe for (bi)sulfites. Currently,
our efforts are directed to determine the metabolic relation of SO2
and biological thiols17e for which the design of probe 3 is going to
be a useful guide.
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We are grateful to TÜBITAK (3501—Career Development
Program (CAREER)/ 114Z806) and BUBAP of Bingöl
University (BAP-834-255-2015, BAP-MMF.2016.00.003 and
BAP-MMF.2017.00.002) for granting this study. We thank the
Central Laboratory of Bingöl University for housing our
research. We are also thankful to Dr. M. Emrullahoğlu, Dr. E.
Karakuş and Dr. N. T. Subaşı for their help in taking some
spectra.
Supplementary Material
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/....
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6
Highlights
 A new water-soluble, highly fluorogenic
3-formylBODIPY dye (3) is synthesized.
 Probe 3 is pH-independent and able to
sense bisulfites in 100% water very
rapidly.
 Probe 3 is able to fluoresce in
adenocarcinoma cells in the presence
of bisulfites.