Iridium(iii) complex-based electrochemiluminescent probe for H2S

Article abstract of DOI:10.1039/c8dt04901g

Since abnormal levels of hydrogen sulphide (H₂S) correlate with various diseases, simple methods for its rapid and sensitive detection are highly required. Herein, we introduce a new electrochemiluminescent probe 1 for H₂S based on a cyclometalated iridium(iii) complex. o-(Azidomethyl)benzoate ester groups on the main ligands of probe 1 react selectively with H₂S, resulting in cascade reactions involving H₂S-mediated reduction and intramolecular cyclization/ester cleavage. With this structural change induced by H₂S, the intrinsic electrochemiluminescence (ECL) of 1 decreased greatly due to the unfavourable electron transfer of a tripropylamine (TPA) radical. Probe 1 showed a high ECL turn-off ratio and good selectivity for H₂S over various anions and biothiols. The sensing mechanism of H₂S was elucidated using¹H NMR spectroscopy and MALDI-TOF mass spectrometry analyses.

Full text of DOI:10.1039/c8dt04901g

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Pleas De ad l too nn oT tr aa nd sj au cs t ti omn sa rgins
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Iridium(III) complex-based electrochemiluminescentDO
p
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4901G
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2
Joonho Park, Taemin Kim, Hoon Jun Kim and Jong-In Hong*
Received 00th January 20xx,
Since abnormal levels of hydrogen sulphide (H
detection are highly required. Herein, we introduce a new electrochemiluminescent probe 1 for H
cyclometalated iridium(III) complex. o-(Azidomethyl)benzoate ester groups on the main ligands of probe 1 react selectively
with H S, resulting in cascade reactions involving H S-mediated reduction and intramolecular cyclization/ester cleavage.
With this structural change induced by H S, the intrinsic electrochemiluminescence (ECL) of 1 decreased greatly due to the
2
S) correlate with various diseases, simple methods for its rapid and sensitive
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
2
S based on a
2
2
2
unfavourable electron transfer of a tripropylamine (TPA) radical. Probe 1 showed high ECL turn-off ratio and good selectivity
1
for H
2
S over various anions and biothiols. The sensing mechanism of H
2
S was elucidated using H NMR spectroscopy and
MALDI-TOF mass spectrometry analyses.
Introduction
2
Hydrogen sulphide (H S) is a toxic gaseous molecule with a
rotten egg smell. However, it is recognized as an important
signalling molecule, along with nitric oxide (NO) and carbon
1
monoxide (CO), because it is involved in various physiological
2
3
4
processes, including angiogenesis, apoptosis, vasodilation,
5
6
7
oxygen sensing, inflammation, and neuromodulation.
Endogenous H
2
S is synthesized from L-cysteine, assisted by
several enzymes like cystathionine β-synthase (CBS),
cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulphur
electrochemical stabilities, high phosphorescence efficiencies,
and easy tunabilities of the emission color by modulating the
8
, 9
transferase (3-MST). In blood plasma, normal concentrations
of H
S range between 10 and 100 μM.2, 10 Abnormal levels of
S are related with several diseases like Alzheimer’s diseases,
2
8, 29
ligand structure.
Herein, we report a new ECL chemodosimetric probe (
S (Scheme 1) based on a cyclometalated iridium(III) complex.
Probe , (AzMB-ppy) Ir(acac), has 2-phenylpyridine-5-yl-2-
azidomethyl)benzoate (AzMB-ppy, AzMB
azidomethyl)benzoate) as the main ligand and acetylacetonate
acac) as the ancillary ligand. Treatment of probe with H
2
1
) for
1
1
H
2
H
2
1
2
13, 14
15
Down’s syndrome,
Therefore, simple, rapid, and reliable detection methods for H
are required for the diagnosis of such diseases. To date, several
S detection methods have been reported, such as gas
diabetes,
and liver cirrhosis.
1
2
2
S
(
(
(
=
H
2
1
2
S
16
17
chromatography,
electrochemical analysis , and metal
nanoparticle sensors , as well as fluorescent and colorimetric
assays. In particular, fluorescent chemodosimeters for H S
2
resulted in chemical reduction of the azide moiety to the
corresponding amine, which underwent simultaneous
intramolecular cyclization and ester cleavage to afford
1
8
19
20
21,22
23,24
were developed based on nucleophilic,
reducing,
or
2
4, 30
compound
2
.
. The structural change of
1
upon chemical
. In order
transition metal-induced precipitation25 properties of sulphide.
Despite their good sensing properties, these probes require
bulky light sources, and thus, cannot be used for point-of-care
testing (POCT).
reaction with H
2
S significantly quenched the ECL of
1
to compare the reaction trend according to the substitution
position of AzMB on the main ligand, we also synthesized
analogues (16
ECL properties with those of probe
ECL probe for H S detection, which utilizes the reducing
property of the sulphide ion.
,
21) of
1
and compared their fluorescence and
In this regard, electrochemiluminescence (ECL), coupled
with chemodosimetry, provides many advantages, allowing
simple analysis in a time- and cost-effective manner. ECL is a
luminescent process involving electron transfer by
electrochemically generated radical species at the surface of
1
. This is the first example of
2
2
6
the working electrode. Since ECL can be generated in the
absence of light sources, it exhibits superior sensitivity without
any background signal, compared to
a
conventional
photoluminescence method. Moreover, it can be miniaturized
26, 27
for POCT applications.
Meanwhile, Ir(III) complexes have
received increasing attention as ECL luminophores with good
Department of Chemistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
Seoul 08826, Korea. E-mail: jihong@snu.ac.kr
†. Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C8DT04901G
3 6
(chloroform = CDCl , dimethyl sulfoxide = DMSO-d ). Matrix-
assisted laser desorption/ionization-time of flight (MALDI-TOF)
mass spectrometry data were obtained using a Bruker Microflex
MALDI-TOF mass spectrometer. Gas chromatography high-
resolution mass spectrometry (GC-HRMS, JEOL, JMS-700) data
were received directly from the National Centre for Inter-
University Research Facilities (NCIRF). Absorption spectra were
recorded on a JASCO V-730 UV-visible spectrophotometer.
Fluorescence emission spectra were recorded on a JASCO FP-
Experimental
Materials and instruments
All the reagents were purchased from Sigma-Aldrich Corp.,
(
(
MO, USA), Alfa Aesar (MA, USA), or Tokyo Chemical Industry
Tokyo, Japan) and used without further purification.
Deuterated solvents were acquired from CIL (Cambridge
Isotopic Laboratories, MA, USA). Merck silica gel 60 F254 on
aluminum foil was used for analytical thin layer
chromatography. SiliaFlash® P60 (230–400 mesh) from
SILICYCLE was used as the stationary phase in chromatographic
6
500 spectrofluorometer. Solutions of probes (1, 16 and 21) for
all the photophysical and electrochemical experiments were
prepared from 2 mM stock solution in DMSO and stored in a
refrigerator for use. NaHS was dissolved in 10 mM HEPES buffer
(pH 7.4) as the source of sulphide ions.
1
13
separation. H NMR and C NMR were recorded on Bruker DPX-
1
1
3
00 (300 MHz for H NMR), Agilent 400-MR (400 MHz for H
13
NMR) and Varian/Oxford As-500 (125 MHz for C NMR)
spectrometers. Chemical shifts (δ) were reported in ppm
2
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DOI: 10.1039/C8DT04901G
Electrochemical and electrochemiluminescent measurements
measurements were referenced with respect to a Ag/Ag+
reference electrode with ferrocene. A Pt working electrode was
polished with 0.05 M alumina (Buehler, IL, USA) on a felt pad
followed by sonication in a 1:1 mixed solution of deionized
water and absolute ethanol for 5 min. The working electrode
was dried using ultra-pure N gas for 1 min. None of the
2
solutions were reused. The reported ECL values were obtained
by averaging the values of at least three repeated experiments
The electrochemical study was conducted using a CH
Instruments 650B Electrochemical Analyzer (CH Instruments,
Inc., TX, USA). Cyclic voltammetry (CV) was applied to individual
solutions in order to investigate electrochemical oxidative and
reductive behavior. The ECL signals were obtained using a low-
voltage photomultiplier tube (PMT) module (H-6780,
Hamamatsu Photonics K. K., Tokyo, Japan) operated at 1.1 V. A
3
in CH CN/DMSO (5:1 v/v).
2
50 μL-sized ECL cell was directly mounted on the PMT module
with home-made support mounts during the experiments. All
the ECL data were collected via simultaneous cyclic
voltammetry (CV). The ECL solutions commonly contained 10
mM TPA (tripropylamine, Sigma-Aldrich, MO, USA) as a co-
reactant and 0.1 M tetrabutylammonium hexafluorophosphate
Synthetic procedures (Schemes 2 and 3)
Synthesis of 4. 2-Bromo-5-hydroxypyridine (1.74 g, 10
mmol) and K CO (2.76 g, 20 mmol) were dissolved in DMF (80
2 3
mL). After stirring at room temperature for 20 min,
iodomethane (0.94 mL, 15 mmol) was added. The mixture was
stirred at room temperature for 6 h. The solvent was removed
under reduced pressure and the reaction mixture was diluted
(TBAPF
6
, TCI) as a supporting electrolyte in acetonitrile (CH
3
CN,
spectroscopy
grade, ACROS). The electrochemical
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Journal Name
with dichloromethane (DCM). The organic phase was washed NMR (300 MHz, DMSO-d
with water and dried over anhydrous Na
6
): δ 10.44 (s, 2H), 8.04 (d, J = 2.3 Hz,
SO4. The volatiles were 2H), 7.93 (d, J = 8.9 Hz, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.39 (dd, J =
View Article Online
DOI: 10.1039/C8DT04901G
2
removed under reduced pressure. The residue was purified by 8.8, 2.4 Hz, 2H), 6.70 (t, J = 7.3 Hz, 2H), 6.53 (t, J = 7.3 Hz, 2H),
silica gel column chromatography using hexane/ethyl acetate 6.02 (d, J = 7.4 Hz, 2H), 5.26 (s, 1H), 1.74 (s, 6H).
(
5:1 v/v) as the eluent to give a pale yellow liquid (1.51 g, 8.03
Synthesis of 1. To a stirred solution of
2
(80 mg, 0.13 mmol)
1
o
mmol, 80%). H NMR (300 MHz, CDCl
3
): δ 8.08 (d, J = 3.0 Hz, 1H), in DCM (5 mL) at 0 C, N,N-diisopropylethylamine (0.1 mL, 0.6
7
.39 (d, J = 8.7 Hz, 1H), 7.12 (dd, J = 8.7, 3.1 Hz, 1H), 3.86 (s, 3H). mmol) was added dropwise via syringe and stirred for 30 min.
Synthesis of 5. A mixture of (940 mg, 5 mmol), In a separate round-bottomed flask, compound 10 (180 mg, 1
phenylboronic acid (671 mg, 5.5 mmol), K CO (2.07 g, 15 mmol) mmol) was refluxed in thionyl chloride (0.37 mL, 5.1 mmol) for
and Pd(PPh (290 mg, 0.25 mmol) in THF (30 mL) and H O (30 2 h and then residual thionyl chloride was removed under
4
2
3
3
)
4
2
mL) was refluxed for 7 h. After cooling to room temperature, reduced pressure to give 2-(azidomethyl)benzoyl chloride. The
the reaction mixture was extracted with DCM. The organic residue dissolved in DCM (5 mL) was added dropwise to a stirred
phase was washed with water and dried over anhydrous solution of
Na SO4. The volatiles were evaporated under reduced pressure. at room temperature for 2 h and extracted with DCM. The
The residue was purified by silica gel column chromatography organic phase was washed with water and dried over anhydrous
2 prepared above. The reaction mixture was stirred
2
with DCM/MeOH (100:1 v/v) as the eluent to give white solid Na
2
SO
4
. The volatiles were evaporated under reduced pressure.
1
(257 mg, 1.39 mmol, 60%). H NMR (300 MHz, CDCl
3
): δ 8.42 (d, The residue was purified by silica gel column chromatography
J = 2.8 Hz, 1H), 7.95 (d, J = 7.2 Hz, 2H), 7.69 (d, J = 8.7 Hz, 1H), with hexane/ethyl acetate (5:1 v/v) as the eluent to give a
1
7
1
.47 (t, J = 7.4 Hz, 2H), 7.41 – 7.36 (m, 1H), 7.31 (d, J = 2.9 Hz, yellow solid. (62 mg, 0.065 mmol, 52%). H NMR (300 MHz,
H), 3.93 (s, 3H).
CDCl
3
): δ 8.55 (d, J = 2.1 Hz, 2H), 8.32 (d, J = 7.7 Hz, 2H), 7.92 (d,
Synthesis of 6. A mixture of
5 (478 mg, 2.58 mmol) and J = 8.9 Hz, 2H), 7.78 – 7.67 (m, 4H), 7.60 (dd, J = 20.7, 7.5 Hz,
iridium chloride hydrate (346 mg, 1.16 mmol) in 2- 6H), 6.87 (t, J = 7.3 Hz, 2H), 6.78 (t, J = 7.2 Hz, 2H), 6.38 (d, J =
ethoxyethanol (30 mL) and H
O (10 mL) was refluxed for 24 h. 7.4 Hz, 2H), 5.31 (s, 1H), 5.00 – 4.85 (m, 4H), 1.84 (s, 6H) ; 13
After cooling to room temperature, water (50 mL) was poured NMR (126 MHz, CDCl ): δ 185.02, 166.60, 164.19, 146.99,
2
C
3
into the reaction mixture. The resulting green precipitate was 145.27, 143.85, 141.38, 138.62, 133.97, 132.99, 131.81, 130.72,
filtered to give a crude cyclometalated Ir(III) chloro-bridged 130.05, 129.33, 128.43, 126.89, 124.04, 120.98, 118.41, 100.80,
+
dimer (426 mg, 0.357 mmol, 62%), which was used in the next 53.12, 28.82; HRMS (FAB , m-NBA): m/z: observed 950.2151
1
+
step without further purification. H NMR (400 MHz, DMSO-d
δ 9.72 (d, J = 2.4 Hz, 2H), 9.24 (d, J = 2.6 Hz, 2H), 8.16 (d, J = 9.0
Hz, 2H), 8.05 (d, J = 8.9 Hz, 2H), 7.76 (dd, J = 9.0, 2.8 Hz, 2H), NBS (1.96 g, 11 mmol) and benzoyl peroxide (48 mg, 0.2 mmol)
6
): (calculated for C43
H
33IrN
8
O
6
[M] 950.2155).
Synthesis of 8.3 Methyl 2-methylbenzoate (1.5 g, 10 mmol),
1
7
2
.69 – 7.62 (m, 4H), 7.58 (d, J = 7.9 Hz, 2H), 6.83 (t, J = 7.5 Hz, were dissolved in CHCl
H), 6.77 (t, J = 7.3 Hz, 2H), 6.68 (t, J = 7.3 Hz, 2H), 6.62 (t, J = 7.4 reaction mixture was cooled to room temperature and the
3
(50 mL) and refluxed for 5 h. The
Hz, 2H), 6.20 (d, J = 7.6 Hz, 2H), 5.60 (d, J = 7.4 Hz, 2H), 3.91 (d, organic phase was washed with saturated aqueous sodium
J = 11.2 Hz, 12H).
thiosulfate solution and dried over anhydrous Na
2 4
SO . The
Synthesis of 7. To a stirred solution of
6 (425 mg, 0.36 mmol) solvent was evaporated under reduced pressure. The residue
o
in DCM (5 mL) at 0 C, BBr
3
(2.14 mL, 1.0 M in DCM) was added was purified by silica gel column chromatography with
dropwise via syringe under N
2
atmosphere. The reaction hexane/acetone (5:1 v/v) as the eluent to give a pale yellow
1
mixture was stirred at room temperature for 24 h, at which time liquid (1.98 g, 8.7 mmol, 87%). H NMR (300 MHz, CDCl
3
): δ 7.99
1
H NMR analysis indicates a complete disappearance of (d, J = 7.9 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.43 – 7.36 (m, 1H), 4.98
methoxy proton at δ 3.91. The reaction was quenched with (s, 2H), 3.97 (s, 3H).
31, 32
water (50 mL) and the resulting green precipitate was filtered
Synthesis of 9.
A mixture of 8 (1.65 g, 7.25 mmol) and
to give a crude cyclometalated Ir(III) chloro-bridged dimer (382 sodium azide (945 mg, 14.5 mmol) in DMF (50 mL) was heated
o
mg, 0.335 mmol, 94%), which was used in the next step without to 70 C with stirring for 24 h. The solvent was then removed
1
further purification. H NMR (300 MHz, DMSO-d
6
): δ 10.80 (s, under reduced pressure and the reaction mixture was diluted
2
8
8
H), 10.53 (s, 2H), 9.49 (d, J = 2.1 Hz, 2H), 9.37 (d, J = 2.2 Hz, 2H), with ethyl acetate. The organic phase was washed with water
.06 (d, J = 8.9 Hz, 2H), 7.96 (d, J = 8.9 Hz, 2H), 7.59 – 7.43 (m, and dried over anhydrous Na SO4. Ethyl acetate was evaporated
H), 6.86 (t, J = 7.4 Hz, 2H), 6.79 – 6.68 (m, 4H), 6.63 (t, J = 7.3 under reduced pressure. The residue was purified by silica gel
column chromatography with hexane/ethyl acetate (7:1 v/v) as
2
Hz, 2H), 6.16 (d, J = 7.5 Hz, 2H), 5.70 (d, J = 7.5 Hz, 2H).
Synthesis of 2. A mixture of
acetylacetone (0.38 mL, 3.7 mmol) and Na
mmol) in 2-ethoxyethanol (15 mL) was heated to 100 C for 1.5 (m, 2H), 7.43 (t, J = 7.5 Hz, 1H), 4.84 (s, 2H), 3.95 (s, 3H).
h. After cooling to room temperature, the reaction mixture was
extracted with ethyl acetate. The organic phase was washed lithium hydroxide monohydrate (1.4 g, 33.3 mmol) in THF (40
with water and dried over anhydrous Na SO . The volatiles were mL) and H O (4 mL) was stirred at room temperature for 24 h.
7
(421 mg, 0.37 mmol), the eluent to give a pale yellow liquid (1.27 g, 6.65 mmol, 92%).
1
2
CO
3
(392 mg, 3.7
3
H NMR (300 MHz, CDCl ): δ 8.04 (d, J = 7.7 Hz, 1H), 7.62 – 7.49
o
31, 32
Synthesis of 10.
A mixture of 9 (1.27 g, 6.65 mmol) and
2
4
2
evaporated under reduced pressure. The residue was triturated The reaction mixture was diluted with water, extracted with
with acetone and hexane, then the resulting green precipitate DCM, which was discarded. The aqueous phase was acidified
1
was filtered to give compound
2
(214 mg, 0.34 mmol, 46%). H with 2 N HCl, and extracted with DCM. The combined organic
4
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Pleas De ad l too nn oT tr aa nd sj au cs t ti omn sa rgins
Journal Name
phases were washed with brine, dried over anhydrous Na
ARTICLE
2
SO4, h. After cooling to room temperature, the reaction mixture was
View Article Online
DOI: 10.1039/C8DT04901G
filtered, and concentrated to give a white solid (1.15 g, 6.50 extracted with ethyl acetate. The organic phase was washed
1
mmol, 98%). H NMR (300 MHz, CDCl
3
): δ 8.19 (d, J = 7.7 Hz, 1H), with water and dried over anhydrous Na
2
SO
4
. The volatiles were
7
.68 – 7.56 (m, 2H), 7.48 (t, J = 7.5 Hz, 1H), 4.91 (s, 2H).
evaporated under reduced pressure. The residue was triturated
33
Synthesis of 3. To a solution of
8
(50 mg, 0.22 mmol) in with acetone and hexane, and then the resulting green
methanol (5 ml), 2 M of ammonia solution in methanol (30 ml) precipitate was filtered to give compound 15 (150 mg, 0.23
1
was added and stirred at room temperature for 3 h. The mmol, 67%). H NMR (500 MHz, DMSO-d6 ) δ 8.60 (d, J = 7.7 Hz,
reaction mixture was concentrated under reduced pressure. 2H), 7.80 (d, J = 4.9 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.00 (dd, J =
The residue was purified by silica gel column chromatography 7.9, 5.6 Hz, 4H), 6.61 (t, J = 7.5 Hz, 2H), 6.43 (t, J = 7.3 Hz, 2H),
with hexane/ethyl acetate (1:1 v/v) as the eluent to give a white 6.05 (d, J = 7.6 Hz, 2H), 5.14 (s, 1H), 1.64 (s, 6H).
1
solid (25 mg, 0.19 mmol, 86%). H NMR (300 MHz, CDCl
(
3
): δ 7.90
Synthesis of 16. To a stirred solution of 1
5
(120 mg, 0.195
o
d, J = 8.1 Hz, 1H), 7.63 – 7.57 (m, 1H), 7.51 (dd, J = 6.9, 4.6 Hz, mmol) in DCM (10 mL) at 0 C, N,N-diisopropylethylamine (0.15
2
H), 7.35 (s, 1H), 4.50 (s, 2H).
Synthesis of 11. 2-Bromo-3-hydroxypyridine (2.61 g, 15 30 min. In a separate round-bottomed flask, compound 10 (270
mmol) and K CO (4.05 g, 30 mmol) were dissolved in DMF (150 mg, 1.5 mmol) was refluxed in thionyl chloride (0.56 mL, 7.7
mL, 0.9 mmol) was added dropwise via syringe and stirred for
2
3
mL). After stirring at room temperature for 30 min, mmol) for 2 h and then residual thionyl chloride was removed
iodomethane (1.40 mL, 22.5 mmol) was added. The mixture was under reduced pressure to give 2-(azidomethyl)benzoyl
stirred at room temperature for 6 h. The solvent was removed chloride. 2-(Azidomethyl)benzoyl chloride dissolved in DCM (10
under reduced pressure and the reaction mixture was diluted mL) was added dropwise to a stirred solution of 15 prepared
with DCM. The organic phase was washed with water and dried above. The reaction mixture was stirred at room temperature
over anhydrous Na
2
SO4. All volatiles were removed under for 2 h and extracted with DCM. The organic phase was washed
SO . The volatiles were
reduced pressure. The residue was purified by silica gel column with water and dried over anhydrous Na
2
4
chromatography using hexane/ethyl acetate (5:1 v/v) as the evaporated under reduced pressure. The residue was purified
1
eluent to give a pale yellow liquid (2.04 g, 10.84 mmol, 72%). H by silica gel column chromatography with hexane/ethyl acetate
NMR (400 MHz, CDCl
J = 8.1, 4.6 Hz, 1H), 7.13 (dd, J = 8.1, 1.4 Hz, 1H), 3.90 (s, 3H).
3
) δ 7.97 (dd, J = 4.6, 1.4 Hz, 1H), 7.21 (dd, (5:1 v/v) as the eluent to give a yellow solid. (70 mg, 0.074
1
mmol, 39%). H NMR (500 MHz, CDCl
3
) δ 8.56 (d, J = 5.5 Hz, 2H),
Synthesis of 12. A mixture of 11 (1.13 g, 6 mmol), 8.47 (d, J = 7.6 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0
phenylboronic acid (805 mg, 6.6 mmol), K CO (2.48 g, 18 mmol) Hz, 2H), 7.68 – 7.64 (m, 4H), 7.59 (d, J = 7.0 Hz, 2H), 7.21 (d, J =
and Pd(PPh (348 mg, 0.18 mmol) in THF (50 mL) and H O (50 7.3 Hz, 2H), 6.65 (dd, J = 15.4, 7.7 Hz, 4H), 6.35 (d, J = 7.4 Hz,
mL) was refluxed for 7 h. After cooling to room temperature, 2H), 5.25 (s, 1H), 4.90 (s, 4H), 1.83 (s, 6H); C NMR (126 MHz,
the reaction mixture was extracted with DCM. The organic CDCl ) δ 184.94, 163.96, 161.37, 149.32, 146.25, 144.18,
phase was washed with water and dried over anhydrous 143.75, 139.21, 134.16, 133.17, 132.47 , 131.79, 130.03, 129.10,
2
3
3
)
4
2
13
3
Na
2
SO4. All volatiles were evaporated under reduced pressure. 128.57, 127.40, 121.15, 120.95, 100.48, 53.11, 30.86, 28.77;
+
The residue was purified by silica gel column chromatography HRMS (FAB , m-NBA): m/z: observed 950.2147 (calculated for
with DCM/MeOH (100:1 v/v) as the eluent to give white solid
+
43
C H
33IrN
8
O
6
[M] 950.2155).
1
(800 mg, 4.32 mmol, 72%). H NMR (400 MHz, CDCl3) δ 8.32 –
Synthesis of 17. A mixture of 2-bromopyridine (1.50 g, 9.49
8
.21 (m, 1H), 7.89 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 mmol), (4-methoxyphenyl)boronic acid (1.59 g, 10.44 mmol),
CO (4.33 g, 31.3 mmol) and Pd(PPh (329 mg, 0.28 mmol) in
Synthesis of 13. A mixture of 12 (500 mg, 2.69 mmol) and THF (50 mL) and H O (50 mL) was refluxed for 7 h. After cooling
iridium chloride hydrate (322 mg, 1.07 mmol) in 2- to room temperature, the reaction mixture was extracted with
ethoxyethanol (30 mL) and H O (10 mL) was refluxed for 24 h. DCM. The organic phase was washed with water and dried over
After cooling to room temperature, water (50 mL) was poured anhydrous Na SO4. The volatiles were evaporated under
(t, J = 7.3 Hz, 1H), 7.11 (dt, J = 8.3, 6.0 Hz, 2H), 3.69 (s, 3H).
K
2
3
3 4
)
2
2
2
into the reaction mixture. The resulting yellow precipitate was reduced pressure. The residue was purified by silica gel column
filtered to give a crude cyclometalated Ir(III) chloro-bridged chromatography with DCM/MeOH (100:1 v/v) as the eluent to
1
dimer (426 mg, 0.36 mmol, 67%), which was used in the next give white solid (1.1 g, 5.93 mmol, 63%). H NMR (300 MHz,
step without further purification.
3
CDCl ) δ 8.67 (d, J = 4.7 Hz, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.84 –
Synthesis of 14. To a stirred solution of 13 (325 mg, 0.27 7.63 (m, 2H), 7.20 (dd, J = 8.5, 3.2 Hz, 1H), 7.02 (d, J = 8.8 Hz,
o
mmol) in DCM (5 mL) at 0 C was added dropwise BBr
1
3
(1.63 mL, 2H), 3.89 (s, 3H).
atmosphere. The reaction Synthesis of 18. A mixture of 17 (200 mg, 1.08 mmol) and
mixture was stirred at room temperature for 24 h. The reaction iridium chloride hydrate (128 mg, 0.43 mmol) in 2-
was quenched with water (50 mL) and the resulting green ethoxyethanol (50 mL) and H O (5 mL) was refluxed for 24 h.
.0 M in DCM) via syringe under N
2
2
precipitate was filtered to give a crude cyclometalated Ir(III) After cooling to room temperature, water (30 mL) was poured
chloro-bridged dimer (249 mg, 0.218 mmol, 80%), which was into the reaction mixture. The resulting yellow precipitate was
used in the next step without further purification.
filtered to give a crude cyclometalated Ir(III) chloro-bridged
Synthesis of 15. A mixture of 14 (400 mg, 0.35 mmol), dimer (153 mg, 0.13 mmol, 59%), which was used in the next
acetylacetone (0.36 mL, 3.5 mmol) and Na
mmol) in 2-ethoxyethanol (15 mL) was heated to 100 C for 1.5
2
CO
3
(371 mg, 3.5 step without further purification.
o
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J. Name., 2013, 00, 1-3 | 5
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Pleas De ad l too nn oT tr aa nd sj au cs t ti omn sa rgins
Page 6 of 9
ARTICLE
Synthesis of 19. To a stirred solution of 18 (100 mg, 0.08
Journal Name
View Article Online
DOI: 10.1039/C8DT04901G
o
mmol) in DCM (5 mL) at 0 C, BBr
3
(0.5 mL,1.0 M in DCM) was
atmosphere. The reaction
added dropwise via syringe under N
2
mixture was stirred at room temperature for 24 h. The reaction
was quenched with water (30 mL) and the resulting green
precipitate was filtered to give a crude cyclometalated Ir(III)
chloro-bridged dimer (75 mg, 0.066 mmol, 78%), which was
used in the next step without further purification.
Synthesis of 20. A mixture of 19 (100 mg, 0.09 mmol),
acetylacetone (0.09 mL, 0.88 mmol) and Na
2
CO
3
(92.9 mg, 0.88
o
mmol) in 2-ethoxyethanol (10 mL) was heated to 100 C for 1.5
h. After cooling to room temperature, the reaction mixture was
extracted with ethyl acetate. The organic phase was washed
2 4
with water and dried over anhydrous Na SO . The volatiles were
evaporated under reduced pressure. The residue was triturated
with acetone and hexane, and then the resulting green
precipitate was filtered to give compound 20 (30 mg, 0.05
1
mmol, 54%). H NMR (400 MHz, DMSO-d
6
) δ 8.89 (s, 2H), 8.24
(d, J = 5.5 Hz, 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.78 (t, J = 7.2 Hz, 2H),
7
2
.46 (d, J = 8.4 Hz, 2H), 7.18 (t, J = 6.2 Hz, 2H), 6.17 (dd, J = 8.4,
.3 Hz, 2H), 5.45 (d, J = 2.3 Hz, 2H), 5.18 (s, 1H), 1.66 (s, 6H).
Synthesis of 21. To a stirred solution of 20 (40 mg, 0.07
o
mmol) in DCM (5 mL) at 0 C, N,N-diisopropylethylamine (0.05
mL, 0.3 mmol) was added dropwise via syringe and stirred for
3
0 min. In a separate round-bottomed flask, compound 10 (90
mg, 0.5 mmol) was refluxed in thionyl chloride (0.19 mL, 2.6
mmol) for 2 h and then residual thionyl chloride was removed
under reduced pressure to give 2-(azidomethyl)benzoyl
chloride. 2-(Azidomethyl)benzoyl chloride dissolved in DCM (5
mL) was added dropwise to a stirred solution of 20 prepared
above. The reaction mixture was stirred at room temperature
for 2 h and extracted with DCM. The organic phase was washed
with water and dried over anhydrous Na SO . The volatiles were
2 4
evaporated under reduced pressure. The residue was purified
by silica gel column chromatography with hexane/ethyl acetate
sulphide probe based on an Ir(III) complex by modulating the
electron density of the pyridyl moiety.
In this regard, we used (hy-ppy)
-phenylpyridine) ( ) as a model compound to apply the on-off
system in ECL for H S. We predicted that functionalization of the
’-position of the pyridyl moiety to the electron-donating
2
Ir(acac) (hy-ppy=3-hydroxy-
6
2
(
5:1 v/v) as the eluent to give a yellow solid. (31 mg, 0.033
2
1
mmol, 47%). H NMR (400 MHz, DMSO-d
6
) δ 8.38 (d, J = 5.4 Hz,
5
2
H), 8.13 (d, J = 8.2 Hz, 2H), 7.93 (dd, J = 16.9, 7.3 Hz, 4H), 7.81
hydroxyl group would destabilize the LUMO, which would stop
the electron transfer from the HOMO of the TPA radical to the
(d, J = 8.5 Hz, 2H), 7.64 (d, J = 6.3 Hz, 2H), 7.53 (d, J = 7.2 Hz, 2H),
7
2
.47 (d, J = 7.4 Hz, 2H), 7.34 (d, J = 6.1 Hz, 2H), 6.74 (dd, J = 8.4,
elevated LUMO level of
withdrawing ester group of probe
and thus facilitate the electron transfer of TPA radical to the
LUMO level of . Therefore, we expected that the ECL intensity
of would be much larger than that of
2
. On the other hand, the electron-
.3 Hz, 2H), 5.83 (d, J = 2.3 Hz, 2H), 5.25 (s, 1H), 4.70 (d, J = 2.9
1
would stabilize the LUMO
+
Hz, 4H), 1.72 (s, 6H); HRMS (FAB , m-NBA): m/z: observed
9
+
50.2152 (calculated for C43
H
33IrN
8
O
6
[M] 950.2155).
1
1
2.
Results and discussion
Mechanism study
1
Design of ECL probe 1
H NMR experiments in DMSO-d were conducted to
6
1
elucidate the sensing mechanism. H NMR spectra (Fig. 1)
revealed that
. The H NMR spectrum of
NMR spectra of
2
Density functional theory (DFT) calculations of (ppy) Ir(acac)
revealed that the highest occupied molecular orbital (HOMO) is
mainly localized on the Ir(III) centre and the phenyl ring of ppy,
while the lowest unoccupied molecular orbital (LUMO) is
localized on the pyridine of ppy.34 A recent study showed that
chemical modifications of the substituent on the metal-
1
was reacted with NaHS (20 eq.) to provide
2
and
1
1
3
1
+ NaHS is the same as a sum of H
2
and
3
. MALDI-TOF mass analysis of probe
1
was also performed following the addition of NaHS in CH
3
CN
(
(
Fig. 2). Before NaHS addition, a peak was observed at 950.9
m/z) corresponding to , and after NaHS addition (20 eq.), a
. These
1
coordinating pyridyl moiety affect the LUMO levels and may
generate significant changes in the emission profiles.35 These
peak was observed at 632.1 (m/z) corresponding to
2
factor inspired us to develop a new chemodosimetric ECL
6
| J. Name., 2012, 00, 1-3
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Page 7 of 9
Pleas De ad l too nn oT tr aa nd sj au cs t ti omn sa rgins
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C8DT04901G
induce significant changes in the phosphorescence of probe
(Fig. S24 and S25).
1
ECL properties of 1
3
We performed ECL measurements in an CH CN/DMSO
solution mixture (5:1 v/v) with 10 μM
.1M TBAPF as a supporting electrolyte. During cyclic
voltammetry (CV), probe itself showed high ECL intensity,
which gradually decreased with addition of NaHS as expected
Fig. 3). A titration curve of was obtained upon the addition
1 and 10 mM TPA with
0
6
1
(
1
of various concentrations of NaHS (Fig. 4), indicating that the
ECL signal decreased linearly over a NaHS concentration
range 40–140 μM (2–14 eq.). The estimated LOD was
calculated to be 11 nM (S/N ratio = 3, n = 3), which is
significantly lower than that obtained in the
phosphorescence method. The selectivity of 1 was also tested
-
-
-
-
-
3
in the presence of 200 μM of anions like F , Cl , Br , I , NO ,
results support the proposed sensing mechanism of
Scheme 1).
2
1 for H S
-
-
2-
2-
2-
2-
2-
-
2 3 4 3 2 3 2 4 2 5
NO , N , SO , SO , S O , S O , S O , SCN , and biothiols
(
like Cys, Hcy, and GSH (Fig. 5). This result showed that these
anions and biothiols could not reduce the ECL intensity except
for I . It is well documented that iodide adsorption onto the
Photophysical properties of 1
We first investigated the UV-vis absorption spectra of
S20). The absorption peak showed a slight change around 260
nm in the presence of NaHS (20 eq.). We also investigated the
-
1
(Fig.
electrode can retard the oxidation processes necessary for
36
ECL.
CV analysis
To confirm our theoretical prediction of the on-off ECL
phosphorescence spectra of probe
1
3
(10 μM in CH CN/DMSO =
5
:1, λex = 380 nm). Probe
1 itself exhibited a strong
phosphorescence emission peak at 538 nm. The addition of
NaHS gradually reduced the emission intensity with a slight
hypsochromic shift to 516 nm over 1 h (Fig. S21). We found a
good linear relationship between the phosphorescence
intensity at 538 nm and the sulphide concentration ranging
from 0 to 200 μM (Fig. S22, Fig. S23). The limit of detection
mechanism, CV analysis was performed and energy levels were
calculated based on the CV measurements (Fig. S26, Table S1).
The LUMO level of
radical so that can generate efficient ECL (Fig. S28). On the
other hand, the LUMO energy level of is slightly higher than
1 is lower than that of HOMO of the TPA
1
2
the HOMO of the TPA radical; thus, ECL could not be generated
due to the unfavourable electron transfer from the TPA radical
(LOD) was estimated to be 102 nM (signal-to-noise (S/N) ratio =
3
, n = 3). Moreover, the phosphorescence of probe was
1
to the LUMO energy level of
2
(Fig. S28). These results suggest
S detection.
selectively quenched by the sulphide (Fig. S21 and S22). In
contrast, no significant emission changes were observed in the
that could be a good on-off ECL probe for H
1
2
-
-
-
-
-
-
presence of any other anions, such as F , Cl , Br , I , NO
3 4 3 2 3 2 4 2 5
3
, NO
2
,
-
2-
2-
2-
2-
2-
-
N , SO , SO , S O , S O , S O , and SCN (Fig. S24 and
Comparison with analogues of probe 1
S25). It is difficult to distinguish biologically significant thiols like
Cys, Hcy, and GSH from sulphide. However, these thiols did not
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J. Name., 2013, 00, 1-3 | 7
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Page 8 of 9
ARTICLE
In order to compare the reaction trend of probes toward
sulphide according the substitution position of the reaction site
on the main ligand, we synthesized probes 16 and 21 (Scheme
Journal Name
Proceedings of the National Academy of ScienceVsie,w2A0rt1ic0le,O1n0lin7e
DOI: 10.1039/C8DT04901G
L. Li, M. Bhatia, Y. Z. Zhu, Y. C. Zhu, R. D. Ramnath, Z. J. Wang,
,
1
0719.
6
F. B. M. Anuar, M. Whiteman, M. Salto-Tellez and P. K. Moore,
FASEB J., 2005, 19, 1196-1198.
3
). First, we investigated the phosphorescence spectra of
probes 16 and 21 (10 μM in CH CN/DMSO = 5:1, λex = 380 nm)
Fig. S29). The PL intensities of 16 and 21 are 0.5 and 0.4 times
3
7
8
K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066.
B. D. Paul and S. H. Snyder, Nat. Rev. Mol. Cell Biol., 2012, 13
,
(
4
99.
T. W. Miller, J. S. Isenberg and D. D. Roberts, Chem. Rev., 2009,
09, 3099-3124.
that of 1, respectively. The phosphorescence spectra of probes
9
1
1
1
1
and 16 showed similar tendency upon reaction with sulphide.
1
The emission intensity of 16 also gradually decreased over 1 h
with the addition of sulphide (Fig. S30 (a)). However, 21 with
green emission (emission maximum at 505 nm) exhibited no
significant changes upon addition of sulphide (Fig. S30 (b)). 16
turned out to be selective for sulphide (Fig. S31).
0 Y.-H. Chen, W.-Z. Yao, B. Geng, Y.-L. Ding, M. Lu, M.-W. Zhao
and C.-S. Tang, Chest, 2005, 128, 3205-3211.
1 K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura,
Biochem. Biophys. Res. Commun., 2002, 293, 1485-1488.
2 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi and B.
Chadefaux-Vekemans, Am. J. Med. Genet., Part A, 2003, 116A
,
ECL measurements of 16 and 21 were performed in a
3
10-311.
mixture of CH
probe (10 mM TPA with 0.1M TBAPF
electrolyte). The ECL intensities of 16 and 21 are 0.1 and 0.3
times that of , respectively (Fig. S32). The ECL intensity of 16
10 µM) decreased with the addition of sulphide (200 μM) while
3
CN and DMSO (v/v 5:1) in the presence of 10 μM 13 Y. Kaneko, Y. Kimura, H. Kimura and I. Niki, Diabetes, 2006, 55
,
1
391.
6
as the supporting
1
1
4 L. Wu, W. Yang, X. Jia, G. Yang, D. Duridanova, K. Cao and R.
Wang, Lab. Invest., 2008, 89, 59.
5 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G.
Rizzo, E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005,
42, 539-548.
1
(
other analytes showed relatively small changes. In contrast, 21
1
1
6 G. A. Cutter and T. J. Oatts, Anal. Chem., 1987, 59, 717-721.
7 B. Spilker, J. Randhahn, H. Grabow, H. Beikirch and P.
Jeroschewski, J. Electroanal. Chem., 2008, 612, 121-130.
8 N. Mahapatra, S. Datta and M. Halder, J. Photochem.
Photobiol., A, 2014, 275, 72-80.
did not show any response to the addition of all the analytes
(Fig. S33). These results show that the substitution position of
the reaction site on the main ligand of Ir(III) complexes is critical
in designing ECL probes.
1
1
2
2
9 F. Yu, P. Li, P. Song, B. Wang, J. Zhao and K. Han, Chem.
Commun., 2012, 48, 2852-2854.
Conclusion
0 J. Bae, M. G. Choi, J. Choi and S.-K. Chang, Dyes Pigm., 2013,
In summary, we have developed an ECL-based
99, 748-752.
chemodosimetric probe
sulphide produced
1
for sulphide. Treatment of
by cascade reactions involving
1 with
1 Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J.
2
Cen and Z. Guo, Angew. Chem., Int. Ed., 2013, 52, 1688-1691.
hydrosulphide-mediated reduction and intramolecular 22 C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R.
Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 50
0327-10329.
3 A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011,
33, 10078-10080.
4 Z. Wu, Z. Li, L. Yang, J. Han and S. Han, Chem. Commun., 2012,
, 10120-10122.
,
cyclization/cleavage, which reduced ECL due to the
unfavourable electron transfer from the TPA radical to the
LUMO of 2.
1
2
2
2
1
4
8
5 K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T.
Komatsu, T. Ueno, T. Terai, H. Kimura and T. Nagano, J. Am.
Chem. Soc., 2011, 133, 18003-18005.
Conflicts of interest
2
2
2
2
6 M. M. Richter, Chem. Rev., 2004, 104, 3003-3036.
7 W. Miao, Chem. Rev., 2008, 108, 2506-2553.
Acknowledgements
8 Y. You and W. Nam, Chem. Soc. Rev., 2012, 41, 7061-7084.
9 Y. Zhou, H. Gao, X. Wang and H. Qi, Inorg. Chem.,, 2015, 54
,
,
1
446-1453.
This work was supported by the NRF grant (No.
3
3
3
3
0 H. A. Henthorn and M. D. Pluth, J. Am. Chem. Soc., 2015, 137
2
018R1A2B2001293) funded by the MSIP.
15330-15336.
1 T. Wada, A. Ohkubo, A. Mochizuki and M. Sekine, Tetrahedron
Lett., 2001, 42, 1069-1072.
2 K. R. Love, R. B. Andrade and P. H. Seeberger, J. Org. Chem.,
Notes and references
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| J. Name., 2012, 00, 1-3
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Dalton Transactions
View Article Online
DOI: 10.1039/C8DT04901G
(
Azmb-ppy) Ir(acac) showed high “turn-off” ECL response for H S.
2
2