Fast responding and selective near-IR Bodipy dye for hydrogen sulfide sensing

Article abstract of DOI:10.1039/c4cc00762j

A Bodipy based, highly selective probe for hydrogen sulfide has been designed, synthesized and demonstrated to detect H₂S in living cells. In this design, the reduction of two arylazido groups change the charge transfer characteristics of the 3,5-distyryl substituents on the Bodipy core, producing a 20 nm bathochromic spectral shift in the absorption band, and quenching of the emission by 85% compared to the original intensity, through photoinduced electron transfer. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00762j

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Fast responding and selective near-IR Bodipy dye
for hydrogen sulfide sensing†
Cite this: Chem. Commun., 2014,
50, 5455
Tugba Ozdemir,a Fazli Sozmen,a Sevcan Mamur,b Turgay Tekinayb and
Engin U. Akkaya*ac
Received 28th January 2014,
Accepted 13th March 2014
DOI: 10.1039/c4cc00762j
www.rsc.org/chemcomm
A Bodipy based, highly selective probe for hydrogen sulfide has
Biological imaging probes working in the near-IR region of
been designed, synthesized and demonstrated to detect H2S in the spectrum have attracted considerable attention in recent
living cells. In this design, the reduction of two arylazido groups years, since the light used for excitation causes much less
change the charge transfer characteristics of the 3,5-distyryl sub- photodamage to cells compared to ultraviolet or visible light
stituents on the Bodipy core, producing a 20 nm bathochromic used in excitation of other probes. In addition, with near IR
spectral shift in the absorption band, and quenching of the emission probes, cell autofluorescence is not an issue. On the other
by 85% compared to the original intensity, through photoinduced hand, Bodipy dyes, which are difluoroboron-chelated dipyrro-
electron transfer.
methene derivatives, seem to be very good choices for design-
ing novel H2S probes due to their desirable properties, such as
high quantum yields, chemical and photochemical stability,
high molar absorption coefficients, and the fact that they
allow straightforward access to near-IR emitting derivatives
(Scheme 1).8
In this work, the Knoevenagel type chemistry has been used
to obtain a near-IR emissive Bodipy derivative 1 with extended
conjugation. Initially, we synthesized Bodipy 5 having three
triethyleneglycol groups. To that end, compounds 3 and 4 were
prepared according to the reports in the literature. Then Bodipy
5 was obtained by the reaction of aldehyde 4 and 3-ethyl-2,4-
dimethylpyrrole. Finally, following recently established proto-
cols, the condensation reaction of p-azidobenzaldehyde 3
yielded the probe 1 (Ff = 0.38, cresyl violet in ethanol as a
reference dye) in the analytically pure state following chromato-
graphic purification. This reaction not only allowed the for-
mation of the target probe in good yields, but also shifted
emission and absorption wavelengths of the probe to the near-
IR region.
H2S probes functioning through the reduction of azido
groups to amines are available in the literature.9 However,
most of them do not respond fast enough. On the other hand,
the probe proposed in this study (compound 1) responds to H2S
with no time delay, practically immediately as the reagents
are mixed.
Upon titration of probe 1 with various concentrations of
Na2S at room temperature in the acetonitrile-buffer mixture
(20 mM HEPES/CH3CN, 40 : 60, v/v, pH = 7.20, 25 1C), a red shift
of about 20 nm was observed in the electronic absorption
spectrum (Fig. 1a); the color change was easily noticeable with
Hydrogen sulfide (H2S) has a characteristic repulsive odor of
rotten eggs, and plays crucial roles in biological processes; as a
result, many groups worldwide are interested in potential
agents that will allow its real-time monitoring.1 Like the other
two gaseous signaling molecules, carbon monoxide (CO)2 and
nitric oxide (NO),3 hydrogen sulfide is a biosynthetic gasotrans-
mitter. These small gaseous molecules are different from the
other messenger molecules regarding their production and
function. Furthermore, because of their small size and charge
neutrality, they can easily pass through the cellular membranes
without affecting any cell signaling response.4 The important
roles of H2S in many metabolic processes, such as cardio-
vascular protection, neuroprotective effect, arrangement of cell
growth, calcium homeostasis and regulation of neurotrans-
mission are well established in the literature.5
H2S is an example of reactive sulfur species, such as thiols,
S-nitrosothiols, sulfenic acids and sulfite, produced enzymati-
cally from cysteine in a series of reactions, mainly catalyzed by
two pyridoxal 50-phosphate-dependent enzymes, cystathionine
b-synthase (CBS) and cystathionine g-lyase (CSE) and indepen-
dently from pyridoxal 50-phosphate by another enzyme,
3-mercaptopyruvate sulfur transferase (3MST).6 Also, elemental
sulfur is another endogenous H2S source.7
a UNAM-National Nanotechnology Research Center, Bilkent University,
06800 Ankara, Turkey
b Life Sciences Practice and Research Center, Gazi University, 06500 Ankara, Turkey
c Department of Chemistry, Bilkent University, 06800 Ankara, Turkey
† Electronic supplementary information (ESI) available: Experimental proce-
dures, structural proofs. See DOI: 10.1039/c4cc00762j
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Fig. 2 Absorption and emission spectra of probe 1 (2.0 mM) in 20 mM
HEPES : CH3CN (40 : 60, v/v, pH = 7.20, 25 1C) in absence and presence of
various anions. Added Na2S concentration is 50 mM and anion concentra-
tions were 100 mM. Excitation wavelength is 650 nm. Experiments were
done in triplicate.
probe 1 offers very good selectivity for sulfide ions (Fig. 2a). No
other species was able to reduce the two azide groups to amine
groups; as a result no changes in the fluorescence emission
spectra were observed. Competition experiments in the presence
of a number of competing anions were also conducted (ESI†),
again corroborating the selectivity of the probe. The electronic
absorption spectra were unchanged as well. We also note that
1000 equivalents of glutathione or cysteine do not cause any
changes in the emission intensity or absorbance spectrum of
probe 1 (Fig. 2).
An NMR titration experiment was performed in order to
investigate the changes in the chemical shifts of the aromatic
protons during the reduction. Fig. 3 shows the partial 1H NMR
spectra of the probe before (Fig. 3a) and after (Fig. 3b) Na2S
addition in CD3CN. The NMR spectrum clearly shows that all
the aromatic protons of probe 1 were shifted upfield due to the
formation of the electron donor amine group as expected. In
addition, mass spectral (HRMS, ESI†) data following sulfide
treatment of probe 1, supports our structure assignment for the
reduction product.
Scheme 1 Synthesis of target probe 1.
We also wanted to demonstrate the utility of probe 1 in
living cells (Fig. 4). Human breast adenocarcinoma cells
(MCF-7) were grown to confluence at 37 1C under 5% CO2 in
Dulbecco’s Modified Eagle Serum (DMEM) containing 1%
penicillin/streptomycin, 10% fetal bovine serum (FBS) and
Fig. 1 Absorption and emission spectra of 1 (2.0 mM) in 20 mM HEPES :
CH3CN (40 : 60, v/v, pH = 7.20, 25 1C) in increasing Na2S concentrations.
Excitation wavelength is 650 nm. Experiments were done in triplicate.
the naked eye. The actual ratio of S2ꢀ/HSꢀH2S concentrations
(speciation) is dictated by the pH of the buffer.
As shown in the fluorescence spectra (Fig. 1b), the emission
of probe 1 was quenched upon increasing Na2S concentrations
at room temperature in 20 mM HEPES/CH3CN solutions
(40 : 60, v/v, pH = 7.20, 25 1C). The detection limit was deter-
mined (ESI†) to be 0.34 mM.
The reduction of the azido group to the amine group (mecha-
nism presented in the ESI†) provides an alternative excited state
process (photoinduced electron transfer, PET), which is respon-
sible for quenching of the fluorescence emission.
The selectivity of probe 1 for the sulfide species was also
investigated. To that end, fluorescence and electronic absorp-
tion spectral data were collected for other reactive sulfur
species (RSS), reactive oxygen species (ROS) and reactive nitro-
gen species (RNS). Emission data provide clear evidence that
Fig. 3 Stacked partial 1H-NMR spectra of probe 1 (A) and the same
spectrum after the addition of Na2S (B) in acetonitrile-D3 at 25 1C.
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Fig. 4 Confocal microscopy images of showing the H2S response of
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In conclusion, we have developed a sensitive H2S detection
probe which operates much faster than the existing H2S probes.
The azide based probe 1 responds to the H2S through reduction
of two azido groups, resulting in instant quenching of the
emission and a noticeable red shift in the absorbance spec-
trum. The highly selective and sensitive nature of probe 1 for
H2S over other reactive species demonstrates the potential
utility of probe 1. Moreover, due to the presence of hydrophilic
moieties in its structure, the probe is water soluble and thus
appropriate for biological applications. Thus, the added H2S
was successfully imaged inside the cells, suggesting the possi-
bility of imaging endogenously produced H2S in real-time and
in the near IR region of the spectrum.
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Chem. Commun., 2014, 50, 5455--5457 | 5457