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이학석사학위논문
이리듐 복합체를 기반으로 하는 전기 화학적
발광 황화수소 센서
Iridium(III) complex-based
electrochemiluminescent probe for H2S
2019년 2월
서울대학교 대학원
화학부 유기화학 전공
박 준 호
이리듐 복합체를 기반으로 하는 전기 화학적 발광 황화수소 센서
Iridium(III) complex-based electrochemiluminescent probe for H2S
지도교수 홍 종 인
이 논문을 이학석사학위 논문으로 제출함.
2019년 2월
서울대학교 대학원 화학부
유기화학전공
박 준 호
박준호 의 이학 석사 학위논문을 인준함.
2019년 2월
위 원 장 이 현 우 (인)
부 위 원 장 홍 종 인 (인)
위 원 민 달 희 (인)
i
ii
Abstract
Iridium(III) complex-based
electrochemiluminescent probe for H2S
Joonho Park
Major in Organic Chemistry
Department of Chemistry
Graduate School
Seoul National University
Hydrogen sulfide (H2S) is a toxic gaseous molecule with an unpleasant rotten
egg smell. As abnormal levels of H2S correlate with various diseases, rapid, sensitive
and simple detection methods are highly required.
Electrochemiluminescence (ECL) based chemosensors provide several
advantages, allowing simple analysis with a time and cost efficient manner. With
these features, it can be miniaturized available for point-of-care-testing (POCT)
detection sensor.
iii
In this study, we designed a new electrochemiluminescent chemodosimetric
probe 1 for H2S based on cyclometalated Iridium(III) complexes. o-
(azidomethyl)benzoyl group on the main ligands reacts selectively with H2S,
inducing cascade reaction of hydrosulfide-mediated reduction and intramolecular
cyclization cleavage. With this structural change by H2S, the ECL intensity of 1
decreased greatly due to the unfavorable electron transfer of TPA radical. Probe 1
showed high turn-off ratio of ECL and good selectivity for H2S over anions and
biothiols. The mechanism study for sensing H2S was performed by using 1H NMR
and MALDI-TOF spectroscopies.
Keywords: Electrochemiluminescence (ECL), Hydrogen sulfide (H2S), ,
Hydrosulfide-mediated reduction, intramolecular cyclization cleavage,
Cyclometalated Ir(III) complex, Point-of-care-testing (POCT)
Student Number: 2017–25981
iv
Contents
Abstract ......................................................................................... ii
Contents ...................................................................................... iv
A. Background ................................................................... 1
A.1. Design principles of fluorescence sensors ................................ 1
A.2. Electrochemiluminescence (ECL) ......................................... 6
A.3. Hydrogen Sulfide (H2S) sensor ............................................ 12
B. Iridium(III) complex-based electrochemiluminecent
probe for H2S .............................................................. 15
B.1. Introduction ......................................................................... 15
B.2. Result and Discussion .......................................................... 17
B.3. Conclusion .......................................................................... 26
B.4. Experimental Section ........................................................... 27
References ........................................................................... 36
국문초록 .............................................................................. 43
감사의 글 ........................................................................... 444
1
A. Background
A.1. Design principles of fluorescence sensors
Luminescence is energy released by a substance in the form of light. Generally,
when an electron in the ground state absorbs light, the excitation of the highest
occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital
(LUMO) occurs, which then relax back to the HOMO, releasing the excess of energy
as light (Fig. 1). Depending on the mode of excitation, there are several types of
luminescence that can be differentiated into bioluminescence, radioluminescence,
chemoluminescence, electrochemiluminescence etc.
The luminescence phenomenon can be categorized as fluorescence and
phosphorescence (Fig. 1). The difference is that the glow of fluorescence stops right
after the source of excitatory radiation is switched off, whereas for phosphorescence,
an afterglow with durations of fractions of a second up to hours can occur.
Figure 1. Jablonski diagram describing the electronic levels of common organic molecules
and possible transitions between different singlet and triplet states.1
2
Figure 2. Schematic diagram of chemosensors based on binding-signaling subunit
approach.
A chemical sensor is a molecule that is used for sensing of an analyte to
produce a detectable change or a signal.2, 3 The signal change of a chemosensor relies
on an interaction occurring at the molecular level, involves monitoring the chemical
activity. Sensing chemical and biochemical analytes with fluorescence is an active
area of research. By modulating fluorescence of fluorophore, it is possible to design
a rational probe for target molecules.
Molecular recognition
There are two types of approach of sensing the analyte. First is
supramolecular-based approach. Binding-signaling subunit approach is mostly based
on the noncovalent supramolecular interaction between the target and recognition
sites of receptor (Fig. 2). If the fluorescence of a receptor changes with a noncovalent
binding of target, the molecular recognition can be possible through the signal
change.
Displacement approach is another supramolecular-based approach (Fig. 3). In
this case, two subunits, a receptor and indicator, form a coordination complex with
a noncovalent supramolecular interaction. When the target analyte is added to the
solution, there is a displacement reaction between indicator and target. If the
3
spectroscopic characters of the two different molecules are different, it can be used
for chemical sensor. The chemical sensor using these supramolecular-based
approaches is called a chemosesnor.
Second is reaction-based approach (Fig. 4). This approach involves the
specific chemical reaction induced by the target. This covalent bonding approach is
usually irreversible, presenting several advantages such as extremely selective
reactivity and direct response possibility to the varying concentration of the analyte.
The chemical sensor using this reaction-based approach is called a chemodosimeter.
Figure 3. Schematic diagram of chemosensors based on displacement approach.
Figure 4. Schematic diagram of chemodosimeter. The target react with the receptor and
remains covalently attached or catalyzes a chemical reaction.
4
Photoinduced electron transfer (PET)
Photoinduced electron transfer is an excited state electron transfer process by
which an excited electron is transferred from donor to acceptor. This process has
been widely studied and used for sensing of cations and anions.3 When the ground
states of electron absorbs the light and excites to the LUMO, it can migrate to an
another part of empty orbital of a molecule (d-PET) or another electron can occupy
the vacant HOMO orbital to block the emission of light (a-PET) (Fig. 5). Because of
this electron transfer, fluorescence quenching occurs with a nonradiative transition
pathway.
Figure 5. Schematic molecular orbital diagram of the excited fluorophore showing PET
process.
5
Föster Resonance Energy Transfer (FRET)
Föster resonance energy transfer, also known as fluorescence resonance
energy transfer is an energy transfer between two light-sensitive molecules. When
donor chromophore absorbs light, making excited state, the energy may transfer to
an acceptor chromophore through nonradiative dipole-dipole interactions (Fig. 6).4,
5 To design a small-molecule based FRET fluorescent probes, some design criteria
should be considered involving energy donor, acceptor and linkers. Also, the
emission spectrum of the donor chromophore should have appropriate overlap with
the absorption spectrum of the acceptor chromophore. The efficiency of FRET is
inversely proportional to the sixth power of the distance between donor and acceptor.
With this feature, FRET is extremely sensitive to the small changes in distance, can
be a good strategy of design the fluorescence sensor.
Along with PET and FRET, excited state intramolecular proton transfer
(ESIPT), intramolecular charge transfer (ICT), formation of excimer, dexter electron
transfer are other sensing mechanism that is also commonly used for design the
fluorescence sensor.
Figure 6. Schematic diagram of the Föster Resonance Energy Transfer (FRET) process.4
6
A.2. Electrochemiluminescence (ECL)
Electrochemiluminescence (ECL) is a luminescent process through the
electron transfer reaction of electrochemically generated radical species at the
surface of working electrode.6 With a simple voltage application, ECL luminophores
such as Ru(bpy)32+ and iridium complexes generate strong light emission at the
working electrode.
ECL method differs from conventional analytical methods. Since the ECL can
be generated in the absence of light sources, it exhibits superior sensitivity without
background signal. Moreover, it can be miniaturized available for point-of-care-
testing (POCT) detection sensor.6, 7 For these reasons, ECL-based analysis have been
used for the detection of some biomolecules in food and water 8, 9, and applied for
detection of various clinically important analytes.10
Reaction mechanism of ECL
One general ECL generation method known as “annihilation” involves
electron-transfer reaction between an oxidized and a reduced species generated by
the alternated pulsing of the potential. Since this process involves both an oxidized
(M+∙) and a reduced (M-∙) form, the emission is only possible when the ECL
luminophore (M) produces sufficiently stable radical ions. The general mechanism
of “annihilation” is outlined below (Fig. 7). 11
M + e- → M-∙ (Reduction)
M - e- → M+∙ (Oxidation)
M-∙ + M+∙ → M* + M (Excited state formation by annihilation)
M* → M + hv (Light emission)
7
Figure 7. The mechanism of annihilation ECL for Ru(bpy)32+.6
Another ECL generation method called “co-reactant ECL” involves electron-
transfer reaction between co-reactant and luminophore with the one directional
potential scanning. A co-reactant is a compound that can produce a reactive
intermediate, such as strong oxidizing or reducing agent by applying the appropriate
potential (Table 1). Since this process undergoes with one directional potential
scanning, it can overcome the limitation of annihilation ECL such as a relatively
small potential window to applicate for bioanalysis.
Table 1. Summary of typical co-reactants and corresponding reactions.12
8
Tri-n-propylamine (TPA) is co-reactant used for “oxidative-reductive”
process. With a oxidative potential, TPA produces the corresponding aminium
radical cation, which rapidly deprotonates to form a highly reductive α-amino alkyl
radical.
TPA – e- → TPA+∙
TPA+∙ → TPA∙ + H+
The oxidation of TPA can occur directly or via “catalytic route” which M+∙
reacts with TPA to form TPA+∙. After the formation of TPA∙ by deprotonation of
TPA+∙, it reacts with M+∙ to form excited state species, M*. The key reaction steps for
ECL luminophore with TPA is as follows (Fig. 8). 11
M - e- → M+∙
TPA - e- → TPA+∙
M+∙ + TPA → M + TPA+∙
TPA+∙ → TPA∙ + H+
M+∙ + TPA∙ → M* + side product
M + TPA∙ → M-∙ + side product
M-∙ + M+∙ → M* + M
M-∙ + TPA+∙ → M* + TPA
M* → M + hv
9
Figure 8. The mechanism of co-reactant ECL for Ru(bpy)32+
and TPA.12
Peroxydisulfate (S2O82-) is a representative co-reactant used for “reductive –
oxidative” process. When S2O82- reduced, the strong oxidant SO4
-∙ is produced then
the electron transfer reaction with ECL luminophore undergoes to form excited state
species, M*. The key reaction steps of reductive-oxidative process with S2O82- is as
follows.6
M + e- → M-∙
S2O82- + e- → SO4
-∙ + SO42-
M-∙ + SO4-∙ → M* + SO4
2-
10
ECL luminophores
A large number of ECL luminophores have been studied in the past several
years. Rubrene, anthracene, perylene diimide, BODIPY (boron dipyrromethene)
are the well-known organic compound as ECL-active fluorophore (Fig. 9).13, 14
Among them, BODIPY dyes are the unique compounds showing good
spectroscopic and electrochemical properties.15 They produce high ECL signals
with considerably good electrochemical stability in both annihiliation and co-
reactant ECL system.
Figure 9. ECL-active organic fluorophores.
The inorganic complexes containing heavy metal such as Ru, Pt, Ir also have
been found to show a great ECL properties. Among them, Ru(bpy)32+ has been
developed as the most important heavy metal complex showing good ECL
behavior (Fig. 10). Ru(bpy)32+ has many advantages such as strong luminescence,
great electrochemical stability and good solubility in both aqueous and non-
aqueous solvents at room temperature. Because of these features, Ru(bpy)23+ has
been widely used in bioanalytical applications.
11
Recently, cyclometalated Ir(III) complexes have drawn significant attention
for the outstanding ECL performances compared to ruthenium complexes.
Especially, by changing the structure of the coordinated ligands, diverse color
emission is possible controlling the HOMO/LUMO energy levels.16 Moreover,
some Ir(III) complexes emitting red light such as (piq)2Ir(acac), (pq)2Ir(acac)
showed higher ECL performances in acetonitrile condition than that of Ru(bpy)32+
(Fig. 10).17
Figure 10. ECL-active inorganic fluorophores.
12
A.3. Hydrogen Sulfide (H2S) sensors
Hydrogen sulfide (H2S) has been regarded as a toxic gas, however, it has an
important role in human body as a cell signaling molecule in many different
physiological systems. Furthermore, a variety of diseases have been associated with
inadequate levels of H2S. Therefore, the development of sensor probes for H2S has
been an active area of research in recent day.
Figure 11. Summary of strategies for the design of H2S fluorescent probes. (a) cleavage of
the R-O bond (b) cleavage of the S-O bond. (c) double bond addition reaction (d) the
reduction of azide (e) the reduction of the nitro group (f) replacement of copper complexes.
13
In general, the design of hydrogen sulfide sensor uses one of the following
mechanisms: (1) cleavage of alcoxyl (R-O) bond; (2) cleavage of the S-O bond; (3)
a double bond addition reaction; (4) the reduction of azide to amine; (5) the reduction
of nitro group to amine; (6) the replacement of copper complexes (Fig. 11).
(1), (2), (3) mechanisms are based on nucleophilic property of thiol. The
thiolysis and double bond addition reaction at physiological pH can be used to detect
H2S over other biothiols because H2S has a pKa value of about 6.918, whereas typical
biothiols in cells (e.g. glutathione, cysteine, homocysteine) have higher pKa values
of about 8.5.19, 20
Based on the reduction ability of H2S, it is possible to design fluorescent
probes using (4) and (5) mechanisms. With the hydrosulfide-mediated reduction of
azide to amine, ESIPT fluorescence control and ICT-based ratiometric emission
change can be used for detection of H2S by controlling the electron-donating ability
of different substituent group.3, 21 In addition, reduction of the nitro group to
corresponding amino group provides another way of designing the new types of
fluorescent probes because the nitro group is considered to be a strong quencher of
fluorophores.22
Complexes of heavy metal and dye have widely been used for sensing
various anions. The recognition mechanism is based on metal-anion affinity. When
anions are added to the probe, the coordinated metal strongly interact with anions
and the detached form of a fluorophore can be detected.23 Paramagnetic metals are
frequently used to regulate fluorescence emissions in chemical and biological
probes.24 Cu2+ is a paramagnetic metal ion that is used mostly for detection of H2S.
14
Figure 12. Examples of organic compound using various sensing strategies for the detection
of hydrogen sulfide. (a) cleavage of the R-O bond25 (b) cleavage of the S-O bond26 (c) double
bond addition reaction27 (d) the reduction of azide28 (e) the reduction of the nitro group29 (f)
replacement of copper complexes30.
15
B. Iridium(III) complex-based electrochemiluminescent
probe for H2S
B.1. Introduction
Hydrogen sulfide (H2S) 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 monoxide (CO),31 because it is involved in various
physiological processes, including angiogenesis,32 apoptosis,33 vasodilation,34
oxygen sensing,35 inflammation,36 and neuromodulation.37 Endogenous H2S is
synthesized from L-cysteine, assisted by several enzymes like cystathionine β-
synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfur
transferase (3-MST).38, 39 In blood plasma, normal concentrations of H2S range
between 10 and 100 μM.32, 40 Abnormal levels of H2S are related with several
diseases like Alzheimer’s diseases,41 Down’s syndrome,42 diabetes,43, 44 and liver
cirrhosis.45 Therefore, simple, rapid, and reliable detection methods for H2S are
required for the diagnosis of such diseases. To date, several H2S detection methods
have been reported, such as gas chromatography,46 electrochemical analysis47, and
metal nanoparticle sensors48, as well as fluorescent49 and colorimetric assays.50 In
particular, fluorescent chemodosimeters for H2S were developed based on
nucleophilic,51, 52 reducing,28, 53 or transition metal-induced precipitation54 properties
of sulfide. Despite their good sensing properties, these probes require bulky light
sources, and thus, cannot be used for point-of-care testing (POCT).
In this regard, electrochemiluminescence (ECL), coupled with
chemodosimetry, provides many advantages, allowing simple analysis in a time- and
16
cost-effective manner. ECL is a luminescent process involving electron transfer by
electrochemically generated radical species at the surface of the working electrode.6
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 for POCT
applications.6, 7 Meanwhile, Ir(III) complexes have received increasing attention as
ECL luminophores with good electrochemical stabilities, high phosphorescence
efficiencies, and easy tunabilities of the emission color by modulating the ligand
structure.55, 56
Herein, we report a new ECL chemodosimetric probe (1) for H2S (Scheme 1)
based on a cyclometalated iridium(III) complex. Probe 1, (AzMB-ppy)2Ir(acac), has
6-phenylpyridine-3-yl 2-(azidomethyl)benzoate (AzMB-ppy) as the main ligand and
acetylacetonate (acac) as the ancillary ligand. Treatment of probe 1 with H2S resulted
in chemical reduction of the azide moiety to the corresponding amine, which
underwent simultaneous intramolecular cyclization and ester cleavage to afford
compound 6.53, 57. The structural change of 1 upon chemical reaction with H2S
significantly quenched the ECL of 1. This is the first example of ECL probe for H2S
detection, which utilizes the reducing property of the sulfide ion.
17
B.2. Result and discussion
Scheme 1. ECL sensing mechanism of H2S of probe 1.
Design of ECL probe 1
Density functional theory (DFT) calculations of (ppy)2Ir(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.58 A recent study showed that chemical
modifications of the substituent on the metal-coordinating pyridyl moiety affect the
LUMO levels and may generate significant changes in the emission profiles.59 These
factor inspired us to develop a new chemodosimetric ECL sulfide probe based on an
Ir(III) complex by modulating the electron density of the pyridyl moiety.
In this regard, we used (hy-ppy)2Ir(acac) (hy-ppy=3-hydroxy-6-
phenylpyridine) (6) as a model compound to apply the on-off system in ECL for H2S.
We predicted that functionalization of the 3’-position of the pyridyl moiety to the
electron-donating hydroxyl group would destabilize the LUMO, which would stop
the electron transfer from the HOMO of the TPA radical to the elevated LUMO level
of 6. On the other hand, the electron-withdrawing ester group of probe 1 would
18
stabilize the LUMO and thus facilitate the electron transfer of TPA radical to the
LUMO level of 6.
Photophysical properties of 1
We first investigated the phosphorescence spectra of probe 1 (10 μM in
CH3CN/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. 13b).
We found a good linear relationship between the phosphorescence intensity at 538
nm and the sulfide concentration ranging from 0 to 200 μM (Fig. 13c and Fig. 13d).
The limit of detection (LOD) was estimated to be 102 nM (signal-to-noise (S/N) ratio
= 3, n = 3). We also investigated the UV-vis absorption spectra of 1 (Fig. 13a). The
absorption peak showed a slight change around 260 nm in the presence of NaHS (20
eq.). Moreover, the phosphorescence of probe 1 was selectively quenched by the
sulfide ion (Fig. 14a and Fig. 14b). In contrast, no significant emission changes were
observed in the presence of any other anion, such as F-, Cl-, Br-, I-, NO3-, NO2
-, N3-,
SO42-, SO3
2-, S2O32-, S2O4
2-, S2O52-, and SCN-. It is difficult to distinguish biologically
significant thiols like Cys, Hcy, and GSH from sulfide. However, these thiols did not
induce significant changes in the phosphorescence of probe 1.
19
Figure 13. (a) UV-vis absorption spectra of 1 (20 μM) before (black) and after (red) the
addition of NaHS (400 μM) in organic solution (CH3CN/DMSO = 5/1) (b) Time-dependent
phosphorescent emission spectra of 1 (10 μM) in the presence of 400 μM NaHS in organic
solution (CH3CN/DMSO = 5/1) (c) Phosphorescent emission spectra of 1 (10 μM) in the
presence of 0-400μM NaHS in organic solution (CH3CN/DMSO = 5/1) (d) PL intensity of
probe 1 (10 μM) at 538nm in the presence of 0-200 μM in organic solution (CH3CN/DMSO
= 5/1).
20
Figure 14. (a) Phosphorescent emission spectra of 1 (10 μM) in the presence of various
analytes (400 μM each, sodium salt) in orgainc solution ( CH3CN/DMSO = 5/1 ) (b) PL
responses (516 nm / 538 nm) of 1 (10 μM) in the presence of various analytes (400 μM each,
sodium salt) in orgainc solution ( CH3CN/DMSO = 5/1 ) (1) Probe 1 only (2) F- (3) Cl- (4)
Br- (5) I- (6) NO3- (7) NO2
- (8) N3- (9) SO4
2- (10) SO32- (11) S2O3
2- (12) S2O42-
(18) S2O52- (14)
SCN- (15) Cys (16) Hcy (17) GSH (18) HS-
21
ECL properties of 1
We performed ECL measurements in an CH3CN/DMSO solution mixture
(5:1 v/v) with 10 μM 1 and 10 mM TPA with 0.1M TBAPF6 as a supporting
electrolyte. During cyclic voltammetry (CV), probe 1 itself showed high ECL
intensity, which gradually decreased with addition of NaHS as expected (Fig. 15a).
A titration curve of 1 was obtained upon the addition of various concentrations of
NaHS (Fig. 15b), 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 in the presence of
200 μM of anions like F-, Cl-, Br-, I-, NO3-, NO2
-, N3-, SO4
2-, SO32-, S2O3
2-, S2O42-,
S2O52-, SCN-, and biothiols like Cys, Hcy, and GSH (Fig. 16). 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 electrode can retard the oxidation
processes necessary for ECL.60
22
Figure 15. (a) ECL intensity of 1 (10 µM) in the presence of various concentrations of sulfide
in CH3CN/DMSO (5:1 v/v, 10 mM TPA, and 0.1 M TBAPF6 as supporting electrolyte) while
the potential is swept at a Pt disk electrode (diameter: 2 mm) in the range of 0–1.1 V (scan
rate: 0.1 V/s). (b) ECL intensity of 1 (10 µM) obtained for ECL titration upon the addition of
various concentration of sulfide in CH3CN/DMSO (5:1 v/v, 10 mM TPA, and 0.1 M TBAPF6
as supporting electrolyte).
Figure 16. (a) ECL responses of Probe 1 (10 μM) in the presence of various analytes (200
μM each, sodium salt) in CH3CN/DMSO (5:1 v/v, 10 mM TPA, and 0.1 M TBAPF6 as
supporting electrolyte) (1) Probe 1 only (2) F- (3) Cl- (4) Br- (5) I- (6) NO3- (7) NO2
- (8) N3-
(9) SO42- (10) SO3
2- (11) S2O32- (12) S2O4
2- (13) S2O52- (14) SCN- (15) Cys (16) Hcy (17)
GSH (18) HS-
23
Figure 17. Comparison of 1H NMR spectra of 1, 1 + NaHS (20 equiv), 6 and 10 (2 mM in
DMSO-d6.)
Figure 18. MALDI-TOF mass spectra of 1 and 1 + NaHS (20 equiv).
24
Mechanism study
1H NMR experiments in DMSO-d6 were conducted to elucidate the sensing
mechanism. 1H NMR spectra (Fig. 17) revealed that 1 was reacted with NaHS (20
eq.) to provide 6 and 10. MALDI-TOF mass analysis of probe 1 was also performed
following the addition of NaHS in CH3CN (Fig. 18). Before NaHS addition, a peak
was observed at 950.9 (m/z) corresponding to 1, and after NaHS addition (20 eq.), a
peak was observed at 632.1 (m/z) corresponding to 6. These results support the
proposed sensing mechanism of 1 for H2S (Scheme 1).
CV analysis
To confirm our theoretical prediction of the on-off ECL mechanism, CV
analysis was performed and energy levels were calculated based on the CV
measurements (Fig. 19, Table 2). The LUMO level of 1 is lower than that of HOMO
of the TPA radical, such that 1 can generate efficient ECL (Fig. 20). On the other
hand, the LUMO energy level of 6 is slightly higher than the HOMO of the TPA
radical; thus, ECL could not be generated due to the unfavourable electron transfer
from the TPA radical to the LUMO energy level of 6 (Fig. 20). These results suggest
that 1 could be a good on-off ECL probe for H2S detection.
25
Figure 19. Cyclic voltammogram of (a) 1, (b) 1 + NaHS in CH3CN.
Table 2. Calculated energy level of 1,6 and TPA radical by CV measurement.
Figure 20. Electronic distributions of probe 1 and 6 from DFT calculations.
26
B.3. Conclusion
In summary, we have developed an ECL-based chemodosimetric probe 1 for
sulfide. Treatment of 1 with sulfide produced 6 by cascade reactions involving
hydrosulfide-mediated reduction and intramolecular cyclization/cleavage, which
reduced ECL due to the unfavourable electron transfer from the TPA radical to the
LUMO of 6.
27
B.4. Experimental Section
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 separation.
1H NMR and 13C NMR were recorded on Bruker DPX-300 (300 MHz for 1H NMR),
Agilent 400-MR (400 MHz for 1H NMR) and Varian/Oxford As-500 (125 MHz for
13C NMR) spectrometers. Chemical shifts (δ) were reported in ppm (chloroform =
CDCl3, dimethyl sulfoxide = DMSO-d6). 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 Center 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-6500 spectrofluorometer. Solutions of probes 1 and 6 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 sulfide ions.
28
Electrochemical and electrochemiluminescent (ECL) measurements
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 250 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
(TBAPF6, TCI) as a supporting electrolyte in acetonitrile (CH3CN, spectroscopy
grade, ACROS). The electrochemical 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 N2 gas for 1 min. None of the solutions were
reused. The reported ECL values were obtained by averaging the values of at least
three repeated experiments in CH3CN/DMSO (5:1 v/v).
29
Scheme 2. (a) Synthesis of Probe 1 (i) K2CO3, CH3I, DMF, r.t; (ii) phenylboronic acid,
Pd(PPh3)4, K2CO3, THF, H2O, reflux; (iii) IrCl3∙xH2O, 2-ethoxyethanol, H2O, reflux; (iv)
BBr3, DCM, 0oC → r.t; (v) acetylacetone, Na2CO3, 2-ethoxyethanol, 100oC; (vi) SOCl2,
reflux; DCM, DIPEA, 0oC → r.t. (b) Synthesis of 2-(azidomethyl)benzoic acid (i) NBS,
benzoyl peroxide, reflux; (ii) NaN3, DMF, 70oC; (iii) LiOH, THF, H2O, r.t; (iv) NH3, MeOH,
r.t; (DMF = N,N-dimethylformamide, THF = tetrahydrofuran, DCM = dichloromethane,
DIPEA = N,N-diisopropylethylamine, NBS = N-bromosuccinimide)
30
Synthesis of Ir(III) complex (Scheme 2)
Synthesis of 2.
2-Bromo-5-hydroxypyridine (1.74 g, 10 mmol) and K2CO3 (2.76 g, 20 mmol)
were dissolved in DMF (80 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 with dichloromethane (DCM). The organic phase was
washed with water and dried over anhydrous Na2SO4. The volatiles were removed
under reduced pressure. The residue was purified by silica gel column
chromatography using hexane/ethyl acetate (5:1 v/v) as the eluent to give a pale
yellow liquid (1.51 g, 8.03 mmol, 80%). 1H NMR (300 MHz, CDCl3): δ 8.08 (d, J =
3.0 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.12 (dd, J = 8.7, 3.1 Hz, 1H), 3.86 (s, 3H).
Synthesis of 3.
A mixture of 2 (940 mg, 5 mmol), phenylboronic acid (671 mg, 5.5 mmol),
K2CO3 (2.07 g, 15 mmol) and Pd(PPh3)4 (290 mg, 0.25 mmol) in THF (30 mL) and
H2O (30 mL) was refluxed for 7 h. After cooling to room temperature, the reaction
mixture was extracted with DCM. The organic phase was washed with water and
dried over anhydrous Na2SO4. The volatiles were evaporated under reduced pressure.
The residue was purified by silica gel column chromatography with DCM/MeOH
(100:1 v/v) as the eluent to give white solid (257 mg, 1.39 mmol, 60%). 1H NMR
(300 MHz, CDCl3): δ 8.42 (d, J = 2.8 Hz, 1H), 7.95 (d, J = 7.2 Hz, 2H), 7.69 (d, J =
8.7 Hz, 1H), 7.47 (t, J = 7.4 Hz, 2H), 7.41 – 7.36 (m, 1H), 7.31 (d, J = 2.9 Hz, 1H),
3.93 (s, 3H).
31
Synthesis of 4.
A mixture of 3 (478 mg, 2.58 mmol) and iridium chloride hydrate (346 mg,
1.16 mmol) in 2-ethoxyethanol (30 mL) and H2O (10 mL) was refluxed for 24 h.
After cooling to room temperature, water (50 mL) was poured into the reaction
mixture. The resulting green precipitate was filtered to give a crude cyclometalated
Ir(III) chloro-bridged dimer (426 mg, 0.357 mmol, 62%), which was used in the next
step without further purification. 1H NMR (400 MHz, DMSO-d6): 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), 7.69 – 7.62 (m, 4H), 7.58 (d, J = 7.9 Hz, 2H), 6.83 (t,
J = 7.5 Hz, 2H), 6.77 (t, J = 7.3 Hz, 2H), 6.68 (t, J = 7.3 Hz, 2H), 6.62 (t, J = 7.4 Hz,
2H), 6.20 (d, J = 7.6 Hz, 2H), 5.60 (d, J = 7.4 Hz, 2H), 3.91 (d, J = 11.2 Hz, 12H).
Synthesis of 5.
To a stirred solution of 4 (425 mg, 0.36 mmol) in DCM (5 mL) at 0oC, BBr3
(2.14 mL,1.0 M in DCM) was added dropwise via syringe under N2 atmosphere. The
reaction mixture was stirred at room temperature for 24 h, at which time 1H NMR
analysis indicates a complete disappearance of methoxy proton at δ 3.91. The
reaction was quenched with water (50 mL) and the resulting green precipitate was
filtered to give a crude cyclometalated Ir(III) chloro-bridged dimer (382 mg, 0.335
mmol, 94%), which was used in the next step without further purification. 1H NMR
(300 MHz, DMSO-d6): δ 10.80 (s, 2H), 10.53 (s, 2H), 9.49 (d, J = 2.1 Hz, 2H), 9.37
(d, J = 2.2 Hz, 2H), 8.06 (d, J = 8.9 Hz, 2H), 7.96 (d, J = 8.9 Hz, 2H), 7.59 – 7.43
(m, 8H), 6.86 (t, J = 7.4 Hz, 2H), 6.79 – 6.68 (m, 4H), 6.63 (t, J = 7.3 Hz, 2H), 6.16
(d, J = 7.5 Hz, 2H), 5.70 (d, J = 7.5 Hz, 2H).
32
Synthesis of 6.
A mixture of 5 (421 mg, 0.37 mmol), acetylacetone (0.38 mL, 3.7 mmol)
and Na2CO3 (392 mg, 3.7 mmol) in 2-ethoxyethanol (15 mL) was heated to 100oC
for 1.5 h. After cooling to room temperature, the reaction mixture was extracted with
ethyl acetate. The organic phase was washed with water and dried over anhydrous
Na2SO4. The volatiles were evaporated under reduced pressure. The residue was
triturated with acetone and hexane, then the resulting green precipitate was filtered
to give compound 6 (214 mg, 0.34 mmol, 46%). 1H NMR (300 MHz, DMSO-d6): δ
10.44 (s, 2H), 8.04 (d, J = 2.3 Hz, 2H), 7.93 (d, J = 8.9 Hz, 2H), 7.48 (d, J = 7.5 Hz,
2H), 7.39 (dd, J = 8.8, 2.4 Hz, 2H), 6.70 (t, J = 7.3 Hz, 2H), 6.53 (t, J = 7.3 Hz, 2H),
6.02 (d, J = 7.4 Hz, 2H), 5.26 (s, 1H), 1.74 (s, 6H).
Synthesis of 1.
To a stirred solution of 6 (80 mg, 0.13 mmol) in DCM (5 mL) at 0oC, N,N-
diisopropylethylamine (0.1 mL, 0.6 mmol) was added dropwise via syringe and
stirred for 30 min. In a separate round-bottomed flask, compound 9 (180 mg, 1 mmol)
was refluxed in thionyl chloride (0.37 mL, 5.1 mmol) for 2 h and then residual
thionyl chloride was removed under reduced pressure to give 2-
(azidomethyl)benzoyl chloride. The residue dissolved in DCM (5 mL) was added
dropwise to a stirred solution of 6 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 Na2SO4. The volatiles were evaporated under
reduced pressure. The residue was purified by silica gel column chromatography
with hexane/ethyl acetate (5:1 v/v) as the eluent to give a yellow solid. (62 mg, 0.065
mmol, 52%). 1H NMR (300 MHz, CDCl3): δ 8.55 (d, J = 2.1 Hz, 2H), 8.32 (d, J =
7.7 Hz, 2H), 7.92 (d, J = 8.9 Hz, 2H), 7.78 – 7.67 (m, 4H), 7.60 (dd, J = 20.7, 7.5
33
Hz, 6H), 6.87 (t, J = 7.3 Hz, 2H), 6.78 (t, J = 7.2 Hz, 2H), 6.38 (d, J = 7.4 Hz, 2H),
5.31 (s, 1H), 5.00 – 4.85 (m, 4H), 1.84 (s, 6H) ; 13C NMR (126 MHz, CDCl3): δ
185.02, 166.60, 164.19, 146.99, 145.27, 143.85, 141.38, 138.62, 133.97, 132.99,
131.81, 130.72, 130.05, 129.33, 128.43, 126.89, 124.04, 120.98, 118.41, 100.80,
53.12, 28.82; HRMS (FAB+, m-NBA): m/z: observed 950.2151 (calculated for
C43H33IrN8O6 [M]+ 950.2155).
Synthesis of 7.61
Methyl 2-methylbenzoate (1.5 g, 10 mmol), NBS (1.96 g, 11 mmol) and
benzoyl peroxide (48 mg, 0.2 mmol) were dissolved in CHCl3 (50 mL) and refluxed
for 5 h. The reaction mixture was cooled to room temperature and the organic phase
was washed with saturated aqueous sodium thiosulfate solution and dried over
anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography with hexane/acetone (5:1 v/v) as
the eluent to give a pale yellow liquid (1.98 g, 8.7 mmol, 87%). 1H NMR (300 MHz,
CDCl3): δ 7.99 (d, J = 7.9 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.43 – 7.36 (m, 1H), 4.98
(s, 2H), 3.97 (s, 3H).
34
Synthesis of 8.61, 62
A mixture of 7 (1.65 g,7.25 mmol) and sodium azide (945 mg,14.5 mmol) in
DMF (50 mL) was heated to 70oC with stirring for 24 h. The solvent was then
removed under reduced pressure and the reaction mixture was diluted with ethyl
acetate. The organic phase was washed with water and dried over anhydrous Na2SO4.
Ethyl acetate was evaporated under reduced pressure. The residue was purified by
silica gel column chromatography with hexane/ethyl acetate (7:1 v/v) as the eluent
to give a pale yellow liquid (1.27 g, 6.65 mmol, 92%). 1H NMR (300 MHz, CDCl3):
δ 8.04 (d, J = 7.7 Hz, 1H), 7.62 – 7.49 (m, 2H), 7.43 (t, J = 7.5 Hz, 1H), 4.84 (s, 2H),
3.95 (s, 3H).
Synthesis of 9.61, 62
A mixture of 8 (1.27 g, 6.65 mmol) and lithium hydroxide monohydrate (1.4
g, 33.3 mmol) in THF (40 mL) and H2O (4 mL) was stirred at room temperature for
24 h. The reaction mixture was diluted with water, extracted with DCM, which was
discarded. The aqueous phase was acidified with 2 N HCl, and extracted with DCM.
The combined organic phases were washed with brine, dried over anhydrous Na2SO4,
filtered, and concentrated to give a white solid (1.15 g, 6.50 mmol, 98%). 1H NMR
(300 MHz, CDCl3): δ 8.19 (d, J = 7.7 Hz, 1H), 7.68 – 7.56 (m, 2H), 7.48 (t, J = 7.5
Hz, 1H), 4.91 (s, 2H).
35
Synthesis of 10.63
To a solution of 7 (50 mg, 0.22 mmol) in MeOH (5 ml), 2 M of ammonia
solution (30 ml, methanol) was added and stirred at room temperature for 3 h. The
reaction mixture was concentrated under reduced pressure. The residue was purified
by silica gel column chromatography with hexane/ethyl acetate (1:1 v/v) as the
eluent to give a white solid (25 mg, 0.19 mmol, 86%). 1H NMR (300 MHz, CDCl3):
δ 7.90 (d, J = 8.1 Hz, 1H), 7.63 – 7.57 (m, 1H), 7.51 (dd, J = 6.9, 4.6 Hz, 2H), 7.35
(s, 1H), 4.50 (s, 2H).
36
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국문초록
황화 이온은 부패한 달걀 냄새를 내며 독성이 있는 가스 물질이다. 체내
비정상적인 황화 이온의 농도는 여러 질병과 연관이 있다고 보고됨에 따라,
체내 황화 이온의 농도를 결정하는 빠르고 정확한 검출 방법이 요구되고 있다.
전기화학적 발광을 기반으로 한 화학 센서는 기존의 분석 방법들에 비해
감도가 높고 측정 방법이 간단하며, 저렴한 검출을 가능케 하는 이점이 있다.
이러한 특징들을 바탕으로, 전기화학발광 센서는 소형화가 가능하며 현장
진단용 설비를 개발하는 잠재적 도구가 될 수 있다.
우리는 이리듐 착물을 이용하여 전기화학발광을 기반으로 한 황화 이온
검출 프로브 (1)를 개발하였다. 프로브 1 은 페닐피리딘에 아지도메틸
벤조일기가 달려있는 주 리간드와 아세틸아세톤의 보조 리간드를 갖는다.
프로브 1 은 황화 이온과 반응 시 아자이드가 환원되면서 분자 내 고리화
반응이 유도된다. 이후 아지도메틸 벤조일기가 이리듐 착물로부터 분리되고,
이에 따라 인광과 전기화학적 발광의 감소가 유도된다. 프로브 1 의
전기화학적 발광은 황화 이온 존재 하에 선형 상관관계를 보이며 감소하였다.
또한, 다른 음이온들이나 바이오티올과는 반응하지 않고, 오직 황화 이온과
선택적으로 반응하여 전기화학 발광을 감소시켰다. 우리는 황화 이온 첨가
전후의 NMR 스펙트럼을 비교하고, MALDI-TOF 질량 분석을 진행함으로써
검출 메커니즘을 입증하였다.
주요어 : 황화 이온, 전기화학발광, 고리형 이리듐 착물, 현장진단용 설비
학번 : 2017-25981
44
감사의 글`
먼저 저와 제 가족을 인도해 주시고 사랑해 주시는 주님께 감사 드립니다.
제가 석사과정을 무사히 마치게 된 것은 모두 주님께서 베풀어 주신 은혜라고
생각합니다.
우선 사랑하는 가족들에게 감사 인사를 전하고 싶습니다. 항상 내 편이
되어주시는 사랑하는 나의 부모님. 엄마 아빠의 사랑과 헌신이 없었다면,
지금까지의 힘든 과정들을 잘 이겨내지 못했을 거예요. 멋지고 반듯한
사람으로 키워주셔서 너무나 감사드립니다. 앞으로는 학생 신분을 벗어난
사회인으로서 엄마, 아빠께 효도하는 자랑스러운 아들이 될게요. 그리고
지금은 미국 유학을 떠난 자랑스러운 나의 형. 어렸을 때부터 항상 챙겨주고
도와주며 이끌어줘서 너무 고마워. 내가 지금까지 올 수 있었던 것은 모두 형의
조언과 도움 때문이라고 생각해. 미국에서의 박사과정 건강하게 잘 마치고
돌아오길 바랄게. 앞으로도 지금처럼 좋은 우애 유지하고 부모님께 효도하며
행복하게 살자.
저의 지도교수님이신 홍종인 교수님께 감사의 말씀을 전하고 싶습니다.
2년간 석사과정 중 학문적인 것뿐만 아니라 영적으로도 많은 가르침을 얻을
수 있었습니다. 앞으로 사회에 나가서도 홍종인 교수님의 제자로써 부끄럽지
않은 사람이 되도록 노력하겠습니다. 학위 논문 심사에 참여해 주시고 조언을
주신 이현우 교수님과 민달희 교수님께도 감사드립니다.
MR lab 식구들에게도 감사의 말씀을 전합니다. 오랜 시간 동안 함께
하면서 많은 정이 들었는데, 막상 헤어진다고 생각하니 아쉬움이 남네요. 지난
45
2년간 너무나 감사했고, 서로의 좋은 기억들만 갖으며 나중에 웃는 모습으로
다시 볼 수 있었으면 좋겠습니다.
마지막으로 대학원 입학부터 졸업까지 함께해준 사랑하는 나의 은나.
언제나 내 편이 돼주고, 좋을 때 슬플 때 항상 함께 해줘서 너무나 고마워.
그동안 서운한 것도 많았을 텐데, 앞으로는 좋은 일만 가득하도록 내가 더
노력할게. 고맙고 사랑해.
이 외에도 나의 모든 친구들과 지인들, 항상 곁에서 힘이 되어줘서
고맙습니다.
2018년 12월
박준호 드림.
