Solvent-controlled novel Cu+ and Cu+/2+ fluorescent "turn-ON" probing

Article abstract of DOI:10.1002/bkcs.10626

Anovel Schiff base probe, carbamoyl salicylimine benzothiazole hydrazine, was prepared and measured for its probing ability. High sensitivity was shown for Cu⁺and Cu²⁺. When solvent polarity was regulated through a combination of H

Full text of DOI:10.1002/bkcs.10626

Article
DOI: 10.1002/bkcs.10626
BULLETIN OF THE
Y. Ha et al.
KOREAN CHEMICAL SOCIETY
+
+/2+
Solvent-controlled Novel Cu andCu
Fluorescent “Turn-ON”Probing
Yonghwang Ha,† Dhiraj P. Murale, Sudesh T. Manjare,
,§
†,§
†,‡,§
Minseong Kim,†
†
†,‡,*
Jeong A. Jeong, and David G. Churchill
†Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701,
Republic of Korea. *E-mail: dchurchill@kaist.ac.kr,
‡
Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), Daejeon 305-701,
Republic of Korea
Received July 16, 2015, Accepted September 29, 2015, Published online December 23, 2015
A novel Schiff base probe, carbamoyl salicylimine benzothiazole hydrazine, was prepared and measured for its
+
2+
probing ability. High sensitivity was shown for Cu and Cu . When solvent polarity was regulated through a
+
+
2+
combination ofH Oand acetonitrile, selective sensing ofCu orthetotal amountofCu andCu waspossible.
2
In addition, a linear hypsochromic fluorescent shift of about 30 nm was shown. A binding stoichiometry of
1
:1 exists at low concentration. Time-dependent emission measurement showed an exponential decay curve
3
+
+
(
τ = 2.99 × 10 s, 10 equiv of Cu ) and a linear decay line (slope = –0.0216, 5 equiv of Cu ). Interference
1
+
2+
experiments in 50% acetonitrile in H O showed that the emission produced by Cu and Cu was not disturbed
2
+
byother metal ionsorby acidityorbasicity. Peroxynitrite changed theemission trends; Cu emission decreased
2
+
(78%) and Cu fluorescenceincreased (28-fold). Biothiols, L-cysteine, DL-homocysteine, reduced glutathione,
+
and N-acetyl-L-cysteine affected the complete reversibility of Cu -induced emission in 50% (v/v) acetonitrile
in H O, relative to partial reversibility of Cu emission. Thus, by regulating the ratio between acetonitrile and
H O, Cu and Cu
2
+
2
+
+/2+
can be probed selectively.
2
Keywords: Solvent polarity, Schiff base, Peroxynitrite, Biothiol, Carbamoyl, Reversibility
Introduction
state, chemical sensing methods are very useful. There are
mainly two strategies for the sensing of copper ions: fluores-
cence and colorimetric assays. In particular, fluorescence
methods have excellent potential to allow researchers to go
deeper with in vivo studies to effect biological metal sensing
with low detection limits.
+
/2+
The determination of Cu
concentrations in biological and
environmental systems has become more and more an impor-
tant diagnostic issue. Copper ion is one of the most impor-
tant metal ions in enzyme-active sites because of its redox
1
–4
3
+
+
properties. Copper, with its two main oxidation states, can
In particular, sensing of Cu is challenging because Cu can
2+
be interchanged via the copper version of “Fenton chemistry.”
This can induce a single electron transfer to biological
substrates and mediate important physiological processes.
However, uncontrolled free metals in biological systems can
give rise to reactive radical species, which can deform many
easily undergo conversion to Cu under ambient conditions
2
via disproportionation. Certain compounds have been known
+
as reliable Cu sensors, which are usually based on chelation
2,10–13
sites involving oxygen, nitrogen, and sulfur
cally engineered proteins.
or geneti-
14
5
kinds of important molecules and may induce ailments such
These probing systems use mainly the ether functional
group combined with sulfur or nitrogen atoms in a closed ring
or open chain-like form.
1
,5–8
as certain neurodegenerative diseases.
It is necessary to develop detection methods for distinguish-
+
2+
2
ingbetween Cu andCu forthediagnosis ofdiseases. Com-
monly, inductively coupled plasma-atomic emission
spectroscopy (ICP-AES), ICP-mass spectrometry (ICP-MS),
or atomic absorption (AA) are used for the analysis of Cu
Here, we report the design of a novel fluorescence “turn-on”
+
2+
molecule for Cu and Cu detection with a carbamoyl salicy-
limine benzothiazole-based molecule in which salicylalde-
hyde is a fluorophore (Scheme 1). When N-dimethyl
carbamoyl chloride was functionalized with the hydroxide
of the salicylaldehyde and then the Schiff base was formed
(Table S1), the ligand did not show fluorescence because of
the existence of a proposed photoinduced electron transfer
9
ions. However, it is very difficult to differentiate between
the different oxidation states of the same metal without spe-
cific pretreatment. For separate detection of each oxidation
(
PET) mechanism; such latency is important in developing
§
The respective present affiliations of Y.H., DPM, and STM are
15,16
efficient“turn-on” species.
expected to take place in the presence of specific metal ions by
blocking PET. Additionally, solvent polarity is important for
Fluorescence “turn-on” isthen
Jungwon University (Chungbuk, Republic of Korea), Korea Institute
of Science and Technology (KIST) (Seoul, Republic of Korea), and
University of Mumbai (Mumbai, India).
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For Compound 2, Compound 1 (1.00 g, 5.18 mmol) was
dissolved in ethanol (10 mL) and added to benzothiazole
hydrazide that was dissolved in H O (10 mL), and refluxed
2
ꢀ
at 90 C for 24 h. A gray precipitate was obtained, which
wasfiltered, washedwithamixtureofdeionizedwaterandeth-
anol (1:1, v/v), dried at room temperature for 24 h, and placed
under vacuum for 24 h. A pale grayish powder was obtained
1
(
8
7
yield, 76%). H-NMR (400 MHz, DMSO), δ 12.29 (br, H ),
1
0
3
4
.20 (s, H ), 7.88 (dd, J
= 7.8 Hz, J
= 1.7 Hz, H14),
1
2
H–H
H–H
3
3
.77 (d, J
= 7.9 Hz, H ), 7.46 (d, J
= 8.0 Hz, H ), 7.41
H–H
6
H–H
5
3
3
4
(ddd, J
= 8.1 Hz, J
.33–7.28 (m, H , H ), 7.18 (dd, J
Hz, H ), 7.11 (td, J
s, H ), 2.94 (s, H ), H-decoupled C-NMR (100 MHz,
= 7.3 Hz, J
= 1.7 Hz, H ),
H–H
H–H
H–H
16
3
4
7
= 8.1, J = 1.2
H–H
= 1.2 Hz, H ), 3.12
5
15
4
H–H
3
4
= 7.6, J
1
7
H–H
1
H–H
13
(
23
24
DMSO) δ 166.92 (C ), 153.70 (C ), 149.43 (C ), 138.50
9
20
18
(
C ), 130.22 (C ), 129.40 (C ), 126.93 (C ), 125.95 (C ),
12 16 2 13 7
1
1
25.77 (C , C ), 125.52 (C ), 123.56 (C ), 121.71 (C ),
4 15 14 17 5
1
21.50 (C ), 118.06 (C ), 36.49 (C ), 36.25 (C ). H-
6
3
3
23
24
1
coupled C-NMR (100 MHz, DMSO), 167.13 (s, C9),
53.86–153.74 (m, C ), 150.35–149.47 (m, C ), 138.61
1
2
0
18
2
C–H C–H
1
(d, JC–H = 170 Hz, C ), 131.02 (dd, J
= 140 Hz, J
12
1
=
8.6 Hz, C ), 129.43 (d, JC–H = 11 Hz, C2),
16
1
1
1
27.18–120.66 (m, C , C , C , C , C , C , C C5),
13 7 4 15 14 17 6,
1
1
18.13 (d, J
= 140 Hz, C ), 36.50 (quartet, J
=
C–H
3
C–H
Scheme 1. Synthesis of probe 2.
1
40 Hz, C ), 36.24 (quartet, J
= 140 Hz, C ), HR-LC/
24
23
C–H
MS: calcd for C H N O S + Na: 363.0892, found: m/z,
17 16 4 2
+
the regulation of molecular fluorescence as well as PET or
3
63.0838 (M + Na) (Figure S1, Supporting Information).
1
7–19
related photomechanisms
the stabilization of ground or excited states of the molecules.
; solvent polarity is related to
20
UV–Visible Absorbance or Fluorescence Measurements.
UV–visible absorbance was measured with the Jasco V–530
instrument. After baseline correction, each sample was ana-
lyzed through a range of wavelengths (200–700 nm). Fluores-
Experimental
cence was checked with
a Shimadzu fluorescence
General Remarks. All reagents of analytical grade were used
as received from Sigma Aldrich (Munich, Germany), TCI
spectrometer (RF–5301 PC model). A quartz cuvette was used
for both UV-visible and fluorescence analysis (path
length 10 mm).
(Tokyo, Japan), and Junsei (Junsei Chemical Co., Ltd.,
Tokyo). For characterization of the probe, one-dimensional
1
13
HR-LC/MS. The mass of the probe was analyzed through
high-resolution liquid chromatography/mass spectrometry
H-NMR, C-NMR, two-dimensional correlation spectros-
copy (COSY), nuclear Overhauser effect spectroscopy
NOESY), heteronuclear single-quantum correlation spec-
(HR-LC/MS, Bruker Daltonik, (Billerica, MA, USA),
(
micro-TOF-QIImodel, Germany). The following were the set-
tings: ion source type, electrospray ionization (ESI); ion polar-
ity, positive mode; nebulizer, 0.4 bar; capillary, 4500 V; dry
troscopy (HSQC), and heteronuclear multiple-bond correla-
tion spectroscopy (HMBC) were performed via a Bruker
Avance 400 MHz spectrometer (Billerica, MA, USA).
Fluorescence analysis was performed with a Shimadzu spec-
trophotometer (RF–5301pc) (Columbia, MD, USA). UV–vis
absorbance was carried out by a Jasco spectroscope (V–530
model) (Jasco, Tokyo, Japan).
ꢀ
heater, 180 C; scan begin, 50 m/z; end plate offset, –500 V;
dry gas, 4.0 L/min; collision cell RF, 250.0 Vpp; solvent,
methanol.
Screening of Metal Ions. Solutions (0.1 M) of various metal
+
2+
2+
2+
2+
2+
3+
2+
+
2
Synthesis of the Probe. Compound 1 was synthesized fol-
ions (Ag , Ca , Cd , Co , Cu , Fe , Fe , Hg , K , Mg
2
1
+
2+
+
2+
2+
lowing a previously reported procedure. Salicylaldehyde
2.00 g, 16.4 mmol) was mixed with N-dimethylcarbamoyl
, Mn , Na , Pb , and Zn , as partnered with perchlorate
+
+
3+
(
anions; Cu and Na as acetates; and Al as chloride) were
prepared in deionized water and added into the probe solution
to make 100 100 μM mu;M as the final concentration. In par-
chloride (3.52 g, 32.8 mmol) in the presence of 1,4-diazabicy-
clo[2.2.2]octane (DABCO, 3.52 g, 32.8 mmol) in DMF
+
2+
2+
2+
(15 mL) under argon at room temperature. After 16 h, distilled
ticular, Cu , Co , Fe , and Mn solutions were prepared
immediately before the reaction and used directly (in less than
1 min) to minimize its transformation into other oxidation
states. A 10-min incubation was performed at room tempera-
ture after mixing of compound 2 with each metal ion, and
water (150 mL) was added and the reaction mixture was stored
ꢀ
at 4 C for 2 h. The solution was extracted with ethyl acetate
(three times). The solvent was evaporated and dried undervac-
uum for 16 h. A yellow oil was obtained (yield 70%).
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+
+/2+
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Solvent-controlled Novel Cu and Cu
Fluorescent
KOREAN CHEMICAL SOCIETY
fluorescence and UV-visible absorbance measurements were
carried out.
Interference Experiments. Compound 2 (10 μM, 1 equiv)
+
2+
underwent reaction with Cu (50 μM, 5 equiv) (or Cu ) fol-
lowed by 10 min incubation at room temperature. Then, each
potentially interfering metal (prepared by the same method
described in the section “Screening of metal ions”) was treated
+
2+
with compound 2 solutions containing Cu (or Cu ). After
0 min incubation, the fluorescence intensity was measured.
1
Job Plotting. A series of 10 solutions whose total concentra-
+
tions of compound 2 and Cu were 22 μM and 50 μM were
prepared from 0.0 to 1.0, in increments of 0.1. After 20 min,
UV-visible absorbance and fluorescence measurements were
obtained.
Reversibility by Biothiols. Compound 2 (10 μM, 1 equiv)
+
2+
and Cu (50 μM, 5 equiv) (or Cu ) were mixed and incubated
for 10 min at room temperature. Then, solutions of biothiols
(
L-cysteine, DL-homocysteine, glutathione (reduced), and N-
acetyl-L-cysteine of concentration 50 μM, 5 equiv) were pre-
pared by dissolving them in distilled water, and adding them
+
2+
into the probe solution containing Cu (or Cu ). After 10 min
of incubation, the fluorescence was measured.
DFTCalculations. Molecularstructures andHOMO(highest
occupied molecular orbital)–LUMO (lowest unoccupied
molecular orbital) level energies were acquired using density
functional theory (DFT) calculations (using Gausian 09.
B3LYP method with 6–31g* basis set and 6–311g* basis
set for Cu only). All calculations were performed in the gas
phase (Table S2).
Figure 1. Emission intensity of compound 2 (10 μM, 1 equiv) with
6 metal ions (10 equiv) in (a) 100% acetonitrile, (b) 50% (v/v) ace-
1
tonitrile in H O. Also see Figures S13-S15.
2
Effect of ROS/RNS. Stock solutions of reactive oxygen spe-
t
cies (ROS) (0.1 M, KO , H O , BuOOH, FeSO + H O ,
2
2
2
4
2 2
t
FeSO + BuOOH, NaOCl) were prepared by careful measure-
10 μM with these three solvent systems. After 20 min, UV–
visible and fluorescence spectra were acquired and analyzed.
As shown in Figure 1, Cu showed a large and good selec-
4
ment and dissolving these in distilled water, and reacting with
the probe solution (Compound 2, 10 μM) to help prepare a
final concentration as 50 μM (5 equiv) for 10 min, followed
by fluorescence and UV–visible absorbance measurements
+
tive “turn-on” emission spectrum, 51-fold relative to probe
emission. In previously reported literature, for sensing of
+
(Figure S7). In addition, peroxynitrite, also a member of the
Cu , chelating cyclic or acyclic thiaza-ligands involving nitro-
2,13
reactive nitrogen species (RNS), was synthesized following
gen or sulfur atoms were required.
This new benzothiazole
2
2
a previously reported procedure. The mixture of NaNO2
0.6 M, 1 mL) and H O (0.7 M, 1 mL) underwent reaction
Schiff base ligand showed good “turn-on” selectivity without
(
ether-type functional groups, as was done earlier.
2
2
2
+
with HCl solution (0.6 M, 1 mL), followed by addition of
Intriguingly, in the mixed 50:50 (v/v) solvent system, Cu
ꢀ
+
NaOH solution (1.5 M, 2 mL) within 5 s at 4 C. The concen-
as well as Cu showed a strong fluorescence turn-on signal,
20-and 24-fold, respectively, to the probe fluorescence inten-
sity at the same wavelength (445 nm). In previous literature,
tration ofperoxynitritewasdetermined byUV–visibleabsorb-
ance using the extinction coefficient value of 1670 M/cm
+
(Figure S8).
there were rare cases for fluorescence turn-on for both Cu
2+
and Cu , attributed to the difficulty of designing a chelating
ligand that satisfies the probe design requirements for both
oxidation states of copper. From our results, it is possible to
detect the total amount, or distribution of both oxidation states,
of copper in the liquid sample in vitro by using the 50:50 sol-
Results and Discussion
Screening of Solutions of 16 Different Metal Ions with UV–
Visible and Fluorescence Spectroscopy in Combination
withAcetonitrile andH O. Sixteenmetalionswerescreened
vent system. However, in 100% H O solvent, no fluorescence
2
2
formeasuring UV–visible absorbance (Figure S2) and fluores-
cence changes with 2 (Figure 1). For fluorescence screening,
three solvent systems were used: 100% acetonitrile, 50% ace-
tonitrile (v/v) in H O, and 100% H O. Compound 2 was dis-
was shown (Figure S3).
Measurement of fluorescence by regulation of solvent vol-
ume ratio between acetonitrile and H O: In a 50:50 (v/v) sol-
vent system, both turn-on fluorescence for Cu and Cu were
2
+
2+
2
2
solved with acetonitrile and diluted to a final concentration of
as shown in Figure 1(b). To achieve further results, we
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+
+/2+
Article
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Fluorescent
KOREAN CHEMICAL SOCIETY
changed the volume ratio of the solvent (= acetonitrile/(aceto-
Interestingly, in less than 400 s, the emission signal at
10 equiv of added Cu reached the maximum intensity and
+
nitrile + H O)), in which solvent polarity regulation can be
2
expected. Fluorescence wavelength and emission intensity
exhibited an exponential decay curve relative to the linear
+
2+
of the probe with Cu or Cu were measured. A linear blue
decay found for 5 equiv loading. The lifetime (τ ) calculated
1
+
2+
3
2
shift of ~32 and 29 nm were shown for Cu and Cu , respec-
tively (Figure 2(a)). This data is consistent with a fluorescent
transition energy gap between the excited state and the ground
state, which gradually increases with increasing concentration
of acetonitrile. In addition, when the X-coordinate is
exchanged for “volume/volume ratio,” linearity was shown
from the time of the highest peak was 2.99 × 10 s (R = 0.999)
+
at 10 equiv of Cu (Figure S5). Some studies have mentioned
+
the disproportion property of Cu in the absence, or presence,
2
3,24
of ligand in polymer chemistry.
They also evaluated the
+
equilibrium constants for disproportionation of Cu in water
= ~ 10 to 10 M ) and acetonitrile (K
10 ) without ligands. However, the disproportionation of
6
7
−1
(K
= 6.3 ×
disp
disp
21
−
(
Figure 2(a)).
+
+
In Figure 2(b), the maximum emission intensity of Cu and
Cu in acetonitrile can proceed efficiently with halide ligands
2
+
Cu showed very different trends except at a low acetonitrile
ratio (0–0.4). After the ratio of 0.5, Cu showed roughly a lin-
ear intensity decrease, whereas Cu displayed a discontinuous
or with certain types of ligand systems: (Me6TREN, tetraden-
2
+
3
tate tertiary amine-based ligand (K = ~1.4 × 10 ). This result
+
supports the exponential decay at 10 equiv. As shown in the
2+
trend. Increasing the ratio of acetonitrile loosened the π–π
interaction and increased the fluorescence intensity. From
interference result of Figure 4(a), Cu showed a quenching
+
2+
effect on Cu -induced emission. Cu , generated by dispro-
+
2+
+
0
.5, Cu showed a relatively high intensity compared to Cu .
portionation of excess Cu in the presence of compound 2,
can quench the emission enhanced by Cu , competitively.
The signal at 5 equiv of Cu showed the most stable prob-
+
+
Importantly, only acetonitrile could discriminate Cu from
Cu with a good separation ratio (31-fold) at 436 nm.
2
+
+
ing, adequate for collecting fluorescence information in this
probing system. However, 5 equiv of Cu showed linearity,
not an exponential signal, over the range from 1039 to
Measurement of Time-Dependent Emission Intensity.
+
+
When continuous fluorescence at different equiv of Cu was
+
measured, ahigher equivofCu showedamorerapidresponse
3
600 s, after a maximum was achieved. Linear fitting gave
than lower equiv values (Figure 3).
the following relation equation: Emission intensity = –
2
0
.0216 × (time) + 181.1 (R = 0.997)” (Figure S5), which
means the rate of fluorescence decrease was constant, and is
seen arising from competitive equilibria that are proposed to
exist between states reflected by the emission created by
+
2+
Cu and the quenching by Cu concentration. At 1 equiv con-
+
centration of Cu , there was no decay during the measuring
time (0–3600 s) (Figure S5). From this result, compound
2
at 5 equiv shows potential for a molecular time sensor for
use as a determinant for the expected disproportionation
+
of Cu .
+
Interference Experiments. In the presence of Cu , the fluo-
rescence for compound 2 was measured with other metal ions
to obtain information about interference (Figure 4; Figure S6).
Figure 2. (a) Shift of wavelength at maximum emission and
(
b) maximum emission intensity of the compound 2 (10 μM, 1 equiv)
+ 2+
with Cu (5 equiv) or Cu (5 equiv) in various solvent mixtures (v/v)
between acetonitrile and H
(
+
2
O. Blue line (compound 2 + Cu ), red line
2+
compound2+Cu ), λex =332 nm, Slitwidth(EX: 3nm, EM:3nm),
Figure3. Time-dependentemissionintensityofcompound2(10μM,
+
MeCN: acetonitrile. Error bars indicate standard deviation, three
repetitions (Figure S4, Supporting Information).
1 equiv) at various equiv of Cu in acetonitrile solvent. λ = 332 nm,
ex
λem = 436 nm, Slit width (EX: 5 nm, EM: 5 nm).
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+
+/2+
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Fluorescent
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2
+
3+
2+
3+
2+
3+
In 100% acetonitrile solvent, Cu , Fe , and Hg showed a
Fe , which means Fe can be differentiated from Fe by
+
2+
+
strong quenching effect (over 96%) on Cu emission. Pb ,
Ag , and Zn decreased the emission intensity slightly to
1%, 28%, and 43%, respectively. Interestingly, signals aris-
ing from Fe and Fe were very different. Fe showed a 2-
fold increase over the control and 50-fold increase over that for
compound 2 in the presence of Cu .
In 50% acetonitrile in H O, almost all metal ions did
not alter Cu or Cu
((b) and (c)). Cu and Cu slightly quenched Cu and Cu
emission values by 48% and 32%, respectively, whereas
0% acetonitrile solvent provided relatively stable probing
+
2+
2
+
2+
2
emission intensity (Figure 4
2+
3+
2+
2+
+
+
2+
5
conditions.
When the effect of Brönsted–Lowry acidity and basicity
+
2+
was investigated on Cu and Cu emission in, e.g., the
in vitro 50:50 (v/v) solvent system, little interference was seen
in the fluorescence signal (Figure S7). Limited interference by
other metal ions, by acidity, or basicity supports that com-
pound 2 can probe the total amount for both oxidation states
+/2+
Cu
in vitro when a 50:50 solvent system is present,
although 48% fluorescence can be generated by interference
2+/+
with addition of Cu
.
+
In addition, the possible interference effects of Cu -based
emission by ROS and RNS was investigated. First, although
ROS did not turn-on the emission intensity of compound 2,
partial interference patterns were present (Figure S8 ((b) and
+
(
c)). This result means that the Cu probing potential is reason-
able in this system despite the presence of ROS. For the
absorbance spectrum data, a new selective peak by compound
2
+ KO was shown at 430 nm, while there were no new char-
2
+
acteristic signals with the presence of compound 2 + Cu +
other ROS. This result suggests that there is a specific and
stronginteraction/reactionbetweentheprobeandO . Second,
−
2
peroxynitrite, a member of RNS, was tested for interference of
+
emission induced by Cu . ONOO showed a 72% decrease on
+
Cu -induced emission intensity, thought to be mainly arising
by the basicity imparted by ONOO in the solvent (Figure S9).
2+
Interestingly, Cu showed a 28-fold emission increase in the
presence of ONOO . Peroxynitrite thus functioned as a toggle
+
2+
for turn-on/off behavior in emission by Cu and Cu in this
probing system (Figure 5).
Determination of Binding Stoichiometry Using Titration
+
and Job Analysis. When the titration of Cu was performed
in100%acetonitrileusing UV–visibleabsorbance, atlowcon-
centration (below 3 equiv) an isosbestic point was observed;
Figure 4. Interference experiments with compound 2 (1 equiv,
1
0 μM) with 15 kinds of metal ions (5 equiv) in the presence of
+
+
(a) Cu (5 equiv) in 100% acetonitrile, (b) Cu (5 equiv) in 50% ace-
2
+
2
tonitrile in H O and (c) Cu (5 equiv) in 50% acetonitrile in
H
(
2
O. (a) λex = 332 nm, slit width (EX: 5 nm, EM: 5 nm), ((b) and
c)) λex = 332 nm, slit width (EX: 3 nm, EM: 3 nm). Measurements
wereacquiredthreetimes, and error barsindicatestandarddeviations.
Figure 5. Profound effect of peroxynitrite on the emission intensity
by presence of Cu and Cu in acetonitrile solvent. λex = 332 nm, slit
width (EX: 5 nm, EM:5 nm) (Figure S9).
+
2+
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+
+/2+
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Solvent-controlled Novel Cu and Cu
Fluorescent
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Figure 6. Titration of compound 2 (10 μM, 1 equiv) with copper(I)
acetate. Emission spectra are shown in the range of 350 to 600 nm;
(
5
inset) emission intensity at 436 nm. λex = 332 nm, slit width (EX:
nm, EM:5 nm), Solvent: acetonitrile. Repeated three times. Error
bar means standard error.
+
Figure 8. Reversibility of compound 2 (10 μM, 1 equiv) with Cu
2+
(
5 equiv) (a) and Cu (5 equiv) (b) by biothiols (5 equiv) in 50%
(
3
v/v) acetonitrile in H O. λ = 332 nm, slit width (EX: 3 nm, EM:
2
ex
nm). Hcy: DL-homocysteine, GSH: glutathione (reduced).
1
:1 binding stoichiometry was predicted at low concentration
because of the design involving one main binding pocket.
However, a multi-binding mode, mainly 1:2 after 60 min of
incubation, can be shown at high concentration, which implies
+
Figure 7. Job plot of compound 2 with Cu . λex = 332 nm, Slit width
EX: 5 nm, EM: 5 nm), Solvent: acetonitrile. Total concentra-
tion: 22 μM.
(
+
that an additional binding site is affected by excess Cu bind-
ing, which in turn we propose as related to the PET
1
5,16,25,27
however, no clear isosbestic point was observed over a value
of4equiv(FigureS10).AsshowninFigure6, thefluorescence
intensity increased in a sigmoidal, not linear, fashion. Interest-
mechanism.
+
2+
Reversibility of Cu and Cu by Biothiols, Signal in 50%
Acetonitrile in H O. In a probing system, reversibility is
important for the recovery of the probe and for continuous
measurement. For reversibility testing, we used the biothiols
L-cysteine, DL-homocysteine (Hcy), reduced glutathione
(GSH), and N-acetyl-L-cysteine.
Some previous studies reported the affinity between metal
ions and R-SH.
(Figure S12). However, in acetonitrile: H O (50:50, v/v),
almost complete reversibility was shown for the Cu -induced
2
+
ingly, a large variationwas shown at 3 equiv of Cu , indicating
a potential breaking point where the structure of the probe may
+
be adapted for sensing of Cu (Figure 6). Job analysis using
fluorescence showed that multiple binding is to be expected,
e.g., 1:2–1:4 (Figure S11). However, the binding ratio at
low concentration near 20 μM showed a 1:1 value as a total
concentration using UV–visible absorbance (Figure 7).
Although there is one main binding pocket observed in the
structure of compound 2 shown in Scheme 1, an additional
28–30
In acetonitrile, there was no reversibility
2
+
fluorescence (Figure 8(a)) by four different biothiol species.
This reflects, first, the robustness of the ligand, and second,
the likely role that coordinated acetonitrile solvent molecules
play, which is important for reversibility; acetonitrile coordi-
+
nitrogen, oxygen, and sulfur atom may bind to Cu allowed
by way of a CAr CAr bond rotation, or direct binding, which
provides multiple binding possibilities between the probe and
metal ions (Figure S16). In particular, the nitrogen atoms in an
imine group or in secondary or tertiary amines are important
sites commonly involved with PET, which is important to
nation seems more inert than H O binding in this probing sys-
2
2+
tem. In addition, Cu -induced emission exhibited partial
reversibility: a near 50% emission decrease could be observed
but still the emission was retained (Figure 8(b)). This may be
2
,25,26
fluorescence turn–OFF behavior”.
For these reasons,
Bull. Korean Chem. Soc. 2016, Vol. 37, 69–76
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
74

BULLETIN OF THE
+
+/2+
Article
Solvent-controlled Novel Cu and Cu
Fluorescent
KOREAN CHEMICAL SOCIETY
+
due to the softness of Cu , which induces better affinity to
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Bull. Korean Chem. Soc. 2016, Vol. 37, 69–76
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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Bull. Korean Chem. Soc. 2016, Vol. 37, 69–76
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www.bkcs.wiley-vch.de
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