Reactivity-based detection of copper(II) ion in water: Oxidative cyclization of azoaromatics as fluorescence turn-on signaling mechanism

Article abstract of DOI:10.1021/ja307316s

An oxidative cyclization reaction transforms nonemissive azoanilines into highly fluorescent benzotriazoles. We have found that introduction of multiple electron-donating amino groups onto a simple o-(phenylazo)aniline platform dramatically accelerates its conversion to the emissive polycyclic product. Notably, this chemistry can be effected by μM-level concentrations of copper(II) ion in water (pH = 6-8) at room temperature to elicit >80-fold enhancement in the green emission at λ_em = 530 nm. Comparative kinetic and electrochemical studies on a series of structural analogues have established that the accelerated reaction rates correlate directly with a systematic cathodic shift in the oxidation onset potential of the azo precursors. In addition, single-crystal X-ray crystallographic analysis on the most reactive derivative revealed the presence of a five-membered ring intramolecular hydrogen-bonding network. An enhanced contribution of the quinoid-type resonance in such conformation apparently facilitates the mechanistically required proton transfer step, which, in conjunction with electron transfer at lower oxidation potential, contributes to a rapid cyclization reaction triggered by copper(II) ion in water.

Full text of DOI:10.1021/ja307316s

Article
pubs.acs.org/JACS
Reactivity-Based Detection of Copper(II) Ion in Water: Oxidative
Cyclization of Azoaromatics as Fluorescence Turn-On Signaling
Mechanism
Junyong Jo, Ho Yong Lee, Wenjun Liu, Andras Olasz, Chun-Hsing Chen, and Dongwhan Lee*
́
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States
S
* Supporting Information
ABSTRACT: An oxidative cyclization reaction transforms
nonemissive azoanilines into highly fluorescent benzotriazoles.
We have found that introduction of multiple electron-donating
amino groups onto a simple o-(phenylazo)aniline platform
dramatically accelerates its conversion to the emissive
polycyclic product. Notably, this chemistry can be effected
by μM-level concentrations of copper(II) ion in water (pH =
6−8) at room temperature to elicit >80-fold enhancement in
the green emission at λem = 530 nm. Comparative kinetic and
electrochemical studies on a series of structural analogues have
established that the accelerated reaction rates correlate directly with a systematic cathodic shift in the oxidation onset potential of
the azo precursors. In addition, single-crystal X-ray crystallographic analysis on the most reactive derivative revealed the presence
of a five-membered ring intramolecular hydrogen-bonding network. An enhanced contribution of the quinoid-type resonance in
such conformation apparently facilitates the mechanistically required proton transfer step, which, in conjunction with electron
transfer at lower oxidation potential, contributes to a rapid cyclization reaction triggered by copper(II) ion in water.
turn-on signaling that is tightly coupled to the selective
recognition and/or triggering event, copper(II)-responsive
probes need to operate in water under ambient conditions
for practical applications in detection and imaging. Macro-
molecular systems built with copper(II)-dependent DNAzymes
meet such requirements.9 Small molecule turn-on fluorescence
probes that are compatible with genuine aqueous environments
are exceedingly rare,8j,k which further highlights ongoing
challenges in this research area.
During our search for a promising model system in which
copper(II) functions as an oxidant to convert nonemissive
reactant to emissive product, we came across the oxidative
cyclization of o-(phenylazo)aniline (1) to benzotriazole (2)
shown in Scheme 1.10 Despite its conceptual appeal of
converting a nonemissive azo dye 1 to a highly fluorescent
product 2, this chemistry typically requires an extended
reaction time in basic organic solvents at elevated temper-
atures,10−12 which significantly diminishes its practical utility as
INTRODUCTION
■
Small molecule fluorophores that target heavy metal ions have
broader impacts on multiple disciplines including biological
imaging, medical diagnostics, industrial process control, and
environmental monitoring.1 As enzyme cofactors and signal
messengers, copper plays many important roles in living
system,2 but a high level of unbound copper ions has
deleterious effects, including oxidative damage or misfolding
of proteins responsible for many neurodegenerative diseases.3
For open-shell metal ions, such as copper(II), multiple
nonemissive de-excitation pathways involving electron transfer
or excited-state energy transfer are typically available to quench
fluorescence.4 As such, existing fluorescent chelates that
respond to copper(II) exploit dual emission from internal
charge transfer (ICT) and shifts in wavelength,5 rather than a
straightforward enhancement in fluorescence intensity upon
metal binding.6
An alternative approach is reactivity-based detection, which
utilizes irreversible chemical reactions to transform nonemissive
precursors to fluorescent products and tracks time-dependent
cumulative effects of metal exposure.7 For example, ring
opening or hydrolysis of certain designer molecules is
accelerated by copper(II) ion functioning as a Lewis acid.8
Within this context, we envisioned that an enhancement in
fluorescence signal that is induced by the redox activity of
copper(II) ion could be an appealing design strategy, which
should not suffer from background signals from potentially
competing Lewis acids in the hydrolysis-based detection
schemes.8 In addition to addressing the requirements for
Scheme 1. Synthetic Routes to Benzotriazoles
Received: July 25, 2012
Published: August 30, 2012
© 2012 American Chemical Society
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a detection method for copper(II) ion under ambient and
biologically relevant conditions.
Taking inspirations from this straightforward functional
group transformation that profoundly impacts molecular
photophysics, we have investigated a series of structural
analogues 4−8 shown in Scheme 2. A strategic placement of
The mechanistic framework outlined in Scheme 3 served as a
good starting point for our structure design to enhance the
chemical reactivity of the parent system 1. Specifically, we
wished to introduce chemical functionalities that can promote
the oxidation, deprotonation, or a combination of both.
RESULTS AND DISCUSSION
■
Scheme 2. Chemical Structures of Functionalized
Azoanilines
Design and Synthesis. A series of o-(phenylazo)aniline
derivatives 4−8 were designed (Scheme 2) and prepared in a
straightforward manner (Scheme 4). Along the series
Scheme 4. Synthetic Routes to Functionalized Azoanilines
a
and Benzotriazoles
electron-donating and water-solubilizing amine groups onto the
simple o-(phenylazo)aniline platform dramatically accelerated
the oxidative cyclization reaction in water, as demonstrated by
the turn-on fluorescence response of 8 toward ppm-level of
copper(II) ions in water at neutral pH. A coherent structure−
reactivity relationship that emerges from a combination of
kinetic, electrochemical, X-ray crystallographic, and computa-
tional studies constitutes the main topic of this paper, the
details of which are provided in the following sections.
a
(a) KOH or NaOH, toluene, Δ. (b) (i) aq HCl/NaNO2, MeOH; (ii)
aq NaOH, MeOH, 0 °C. (c) Cu(OAc)2, MeCN or THF, pyridine, Δ.
BACKGROUND: FROM AZO TO TRIAZOLE
■
The thermal decomposition of o-azidoazobenzene 3 is one of
the oldest known synthetic routes to benzotriazole 2 and its
analogues (Scheme 1),13,14 which was reported as early as in
1887.15 More conveniently, 2 is prepared by the oxidation of
azoaniline 1 (Scheme 1), a synthetically more accessible
precursor that can be subjected to large-scale laboratory
manipulations. As shown in Scheme 3, oxidative cyclization
5 → 6 → 7, an increasing number of electron-donating
amine groups were installed either in the upper (= phenyl) or
lower (= aniline) ring of the o-(phenylazo)aniline platform of
the benchmark system 4. For the compound 8, two hydroxyl
groups were introduced as part of N-alkyl tethers to enhance
the water solubility of 7, the most electron-rich system.
As summarized in Scheme 4, simple condensation reactions
between para-substituted nitrosobenzenes and o-phenylenedi-
amine produced 4 and 5. Compounds 6−8 were prepared in
high yields (88−95%) from m-phenylenediamine and the
corresponding diazonium salts, generated in situ under standard
azo coupling conditions. Preparative-scale transformation of 4−
8 to the corresponding benzotriazole products 10−14 (Scheme
4) was achieved by using copper(II) as oxidant and pyridine as
base.
a
Scheme 3. Oxidative Cyclization of Azoaniline
Photophysical Properties. With a series of functionalized
benzotriazoles 10−13 (Scheme 4) in hand, we investigated
their photophysical properties. Unlike their precursors 4−7,
which are nonemissive as typical azo dyes, photoexcited 10−13
are highly fluorescent (Figure 1). Notably, the fluorescence
quantum yield (= ΦF) of 10 is essentially 100% (in MeCN)
when excited at λ = 320 nm. Substitution on the benzotriazole
core and/or the N-aryl ring did not significantly compromise
the emission efficiency, with ΦF = 26−42% determined for 11−
13 in MeCN (Table 1).
The fluorescence spectra of 10−13 in MeCN (Figure 1a)
also revealed systematic red-shifts (Δλ = 70 nm) in the
emission energy along the series 10 → 12 → 13 → 11. This
empirical trend apparently reflects the charge-separated nature
of the excited states,19 which involve electron-deficient
benzotrizole core and electron-rich N-aryl substituent part of
the molecule. As can be anticipated from the chemical
a
For clarity, coordination of metal ions to the amino/azo N-donor is
not shown. Alternative sequences of electron- and proton-transfer (ET
and PT, respectively) steps could also be considered that avoid the
development of highly charged intermediates in the net removal of
2H+/2e− from 1 to form 2.
of 1 proceeds via a net loss of two protons and two electrons,
and is driven by the stability of the newly formed
heteroaromatic triazole ring of 2. With Pb(IV) or Cu(II) as
an oxidant, the 1 → 2 conversion is postulated to involve the
areneamino radical intermediate 9 (Scheme 3),11,16,17 which
undergoes intramolecular N−N coupling rather than inter-
molecular dimerization. Coordination of metal ions to the N-
donor group assists the consecutive deprotonation/oxidation
steps as the entry point to this chemistry.18
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Figure 2. (a) Emission spectra (λexc = 380 nm) of 7 in MeCN mixed
with 3% (v/v) H2O, prior to (black dotted line) and after (blue solid
line) addition of Cu(OAc)2 (100 equiv) at T = 298 K. (b) Normalized
fluorescence spectra of the (i) 7 + Cu(OAc)2 reaction mixture (blue
solid line) and (ii) 13 (blue dotted line). (c) X-ray structure of 13 as
ORTEP diagrams with thermal ellipsoids at 50% probability.
Figure 1. (a) Normalized emission spectra of 10−13 in MeCN. (b)
Polarity-dependent shifts in the emission maximum of 13 plotted as
wavenumber (cm−1) vs ET(30) for various solvents including hexane
(1), benzene (2), toluene (3), THF (4), CHCl3 (5), CH2Cl2 (6),
MeCN (7), and DMSO (8). T = 298 K.
the product 13 was monitored by time-dependent enhance-
ment in the fluorescence intensity at λem = 480 nm (= ΔI480 nm
;
λexc = 380 nm) in MeCN with 3% (v/v) H2O at T = 298 K.
The exponential increase in ΔI480 nm as a function of time was
fitted using eq 1 to determine the pseudo-first-order rate
constant k′ (Figures 3a and S4):
Table 1. Photophysical Properties of Functionalized
Benzotriazoles
ΔI
I
= 1 − e−k′t
(1)
a
compound
λmax,abs (nm)
λmax,em (nm)
ΦF (%)
10
11
12
13
310
370
360
380
430
500
455
480
100
42
26
39
a
In MeCN at T = 298 K.
structures (Scheme 4), such charge-transfer (CT)-type
emission should be most pronounced for 11. Structurally
related 13 also displays strong solvent-dependent fluorescence
spectra (Figure S1) with an excellent linear correlation between
the emission energy and ET(30) values (Figure 1b).20 On the
other hand, the emission from the reference system 10 remains
largely insensitive to solvent polarity (Figure S2). With
appropriate functionalization, the charge-separated excited-
states of N-aryl benzotriazoles should thus produce longer-
wavelength emission in polar aqueous environments (vide
infra), with large Stokes shifts (Table 1; Figure S3) to minimize
fluorescence reabsorption or self-quenching.
Fluorescence Kinetic Studies: Structure−Reactivity
Relationships. In order to explore the functional role of the
electron-donating aryl substituents in the reaction mechanism
shown in Scheme 3, we decided to carry out comparative
kinetic studies on the oxidative cyclization of 4−7. As shown in
Figure 2a, the reaction between the nonemissive azoaniline 7
and Cu(OAc)2 in MeCN proceeds at room temperature with
no added base to furnish a highly fluorescent product, the
emission spectrum of which is essentially superimposable with
that of the benzotriazole 13 (Figure 2b), prepared and
characterized independently (Figure 2c).
Figure 3. (a) Time-dependent changes in the fluorescence intensity at
λ = 480 nm (λexc = 380 nm) observed for the reaction between 7 (2.0
μM) and Cu2+ (1.8 mM) in MeCN mixed with 3% (v/v) H2O at T =
298 K. The gray curve overlaid on the experimental data points is a
theoretical fit generated using k′ = 4.07 × 10−2 s−1 and eq 1. (b) A plot
of k′ (=k[Cu2+]0) vs [Cu2+]0 to determine the second-order rate
constant k = 23 1 M−1 s−1.
A linear dependence of k′ (= k[Cu2+]o; eq 1) on [Cu2+]o
(Figure 3b) established that the reaction proceeds via
bimolecular rate-limiting step with first-order in both copper-
(II) ion and azo dye, and the second-order rate constant of k =
23 1 M−1 s−1. Under similar conditions, the compounds 5
and 6 also react with copper(II) in second-order fashion to
furnish 11 and 12, respectively (Figures S5 and S6), but their
formation kinetics are significantly slower. Specifically, the
second-order rate constants of k = 0.54
0.05 M−1 s−1
0.06
determined for the conversion of 5 → 11 and 0.73
M−1 s−1 for the conversion of 6 → 12 are ca. 30−40-times
slower than that of 7 at T = 298 K. The simple azoaniline
reference system 4 does not even react with copper(II) at room
temperature. Our kinetic studies have thus established the
Under pseudo-first-order reaction conditions (>300 equiv of
[Cu2+] with respect to the azo precursor 7), the formation of
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reactivity order of 7 > 6 ∼ 5 ≫ 4, which correlates directly with
the number of amine groups that are introduced to the
common o-(phenylazo)aniline platform (Scheme 2).
Table 2. Oxidation Potential and Rate Constant of Oxidative
Cyclization of Azoanilines (T = 298 K)
a
compound
Eox/onset (V)
k2 (M−1 s−1
)
Electrochemical Studies: Structure−Property Rela-
tionships. As depicted in Scheme 3, oxidative cyclization of
azoanilines is postulated to proceed via consecutive PT−ET
steps. Our kinetic studies described in the previous section have
established that electron-rich azoanilines display faster response
to copper(II) ions in the bimolecular reaction. In order to
obtain a quantitative understanding of the relationship between
the thermodynamics of oxidation and the kinetics of ring
closure, we carried out electrochemical studies on 4−7.
Under typical conditions used for cyclic voltammetry (CV),
the unsubstituted azoaniline 4 showed an irreversible oxidation
wave with the onset potential of Eox/onset = 0.55 V (Figure 4).21
b
4
5
6
7
0.55
0.20
−
0.54 0.05
0.21
0.73 0.06
−0.06
23
1
a
b
Onset oxidation potential referenced to Fc/Fc+. No reaction.
ET, rather than PT. This interpretation is based on the
assumption that the trend observed for electrochemically
determined Eox/onset values (Figure 4) reflects the facility with
which inner-sphere ET occurs from the amine/amide group to
the copper(II) ion functioning as a chemical oxidant.
X-ray Crystallographic Studies: Functional Role of π-
Conjugation and Hydrogen Bonds. An increasing number
of electron-donating groups enhance the chemical reactivity of
azoanilines toward oxidative cyclization. While this intuitive
structure−reactivity relationship was validated and reinforced
by a combination of kinetic and electrochemical studies
described in previous sections, we wondered whether such
redox argument could solely account for the remarkable
reactivity of 7 compared with its structural analogues 4−6. X-
ray crystallographic studies on 7 provided significant insights
into this question.
A single crystal of 7 was obtained by vapor diffusion of
pentane into a methanol solution and subjected to crystallo-
graphic structure analysis. Intriguingly, 7 exists as two rotational
isomers 7A and 7B (Figure 5a) in the solid-state, which are
Figure 4. CVs of 4−7 (sample concentration = 3.5 mM) in CH2Cl2
n
with Bu4NPF6 (0.1 M) as supporting electrolyte (scan rate = 100
mV/s; T = 298 K). For each CV, the oxidation onset potential is
indicated by a vertical arrow.
Figure 5. (a) X-ray structure of 7 as ORTEP diagrams with 50%
thermal ellipsoids. The two conformations 7A (site occupancy = 32%)
and 7B (site occupancy = 68%) constituting the disorder are shown in
(b).
Introduction of additional amine group, either in the upper ring
of 5 (Eox/onset = 0.20 V) or in the lower ring of 6 (Eox/onset
=
0.21 V), resulted in significant cathodic shifts of the oxidation
potential (Figures 4). With both the upper and lower rings
functionalized with amine groups, compound 7 has the most
cathodically shifted Eox/onset value of −0.06 V. A systematic
cathodic shift in the oxidation onset potentials of 7 > 6 ∼ 5 > 4
(Figure 4; Table 2) observed here correlates nicely with the
second-order rate constants for the oxidative cyclization of 7 >
6 ∼ 5 ≫ 4 (Figures 3 and S4−S6; Table 2).
The reaction mechanism postulated in Scheme 3 most likely
requires coordination of the copper(II) ion to the amine/amide
nitrogen atom to facilitate both PT and ET, with the possibility
of azo nitrogen atom functioning as an additional ligand to
assist metal binding.22 Our electrochemical studies now suggest
that the rate-limiting step in this cyclization reaction involves
related by ca. 180° rotation around the C−N bond. In the
rotamer 7A (Figure 5b), the Nα atom of the azo group is
engaged in a typical six-membered intramolecular hydrogen
bonding with the ortho-NH2 group (N···N = 2.884 Å).23,24 The
major (68%) component of the solid-state structure of 7,
however, is represented by the other rotamer 7B (Figure 5b),
which has a rather unusual five-membered intramolecular
hydrogen bonding between the Nβ atom of the azo group and
the amine N−H group (N···N = 2.603 Å).25 An equilibrium
between six- and five-membered “chelate” rings through N−
H···N hydrogen bond was suggested by UV−vis spectroscopic
studies on a structurally related molecule.26,27 Prior to our
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work, however, no structural evidence has been reported to
support this notion.
The solid-state structure of the azo compound 7 thus reflects
a conformational equilibrium between 7A and 7B with
contributions from the hydrazone resonance structure (Scheme
5). In order to confirm that such conformational bistability has
intramolecular hydrogen bond might thus contribute to the
remarkable reactivity of 7 relative to its analogues 4−6.
Fluorescence Turn-On Detection of Copper(II) Ion in
Water. With a better understanding of the structure−
property−reactivity relationships obtained for the azo pre-
cursors 4−7 and their cyclization products 10−13, we
investigated the scope of this chemistry in physiologically
relevant environment. Using 7 as template, 8 was designed to
enhance the water solubility of the parent system but without
compromising its high reactivity. With the key structural
features of amine-functionalized azoaniline platform retained,
installation of hydroxyl groups as part of the N-alkyl tethers
rendered 8 soluble up to 30 μM in buffered (pH = 7) water
(Figure S7). A linear relationship between the concentration
and the absorbance indicated no intermolecular association
under this condition.
Scheme 5. Conformational Bistability of 7A and 7B through
(i) Formation of the Six- vs Five-Membered Hydrogen Bond
and (ii) Contribution of the Zwitterionic Resonance
Structure
Upon exposure to copper(II) in water (pH = 7.0; HEPES, 50
mM), the strong absorption of 8 at λmax,abs = 475 nm, which is
characteristic of its azo chromophore, rapidly lost its intensity.
This process is accompanied by concomitant build-up of a new
blue-shifted feature at λmax,abs = 375 nm from the cyclized
product 14 (Figure 7a). This metal-induced chemical trans-
formation could be monitored in a straightforward manner by a
large (>80-fold) enhancement in the fluorescence intensity at
λmax,em = 530 nm, which is discernible even by naked eyes
(Figure 7b). No interference, such as paramagnetic quenching,
its origin in the intrinsic thermodynamic properties of the
molecule, we carried out geometry optimization at the MP2
level of theory (with a triple-ζ basis set) on the simplified
models 7A′ and 7B′, in which the p-diethylamino group of the
parent system has been replaced with a simpler p-
dimethylamino moiety.
As shown in Figure 6, the energy-minimized structures 7A′
and 7B′ are essentially superimposable onto the crystallo-
Figure 6. An overlay of the X-ray structures of (a) 7A and (b) 7B (in
black) with the corresponding MP2/TZVP models 7A′ and 7B′ (in
gray), respectively.
graphically determined atomic coordinates of 7A and 7B,
respectively. More importantly, the energy difference between
7A′ and 7B′ is as small as 0.6 kcal mol−1.28
The coexistence of the two essentially isoenergetic rotamers
of 7 presumably reflects a fine balance between (i) steric
preference for the unstrained six-membered hydrogen bond of
7A with the N−H···Nα contact and (ii) electronic preference
for the five-membered hydrogen bonding of 7B with the N−
H···Nβ contact to stabilize the zwitterionic resonance structure.
We postulate that this five-membered hydrogen bond should
allow the azo Nβ position to interact directly with the ortho-
amino group and activate its N−H bond for deprotonation and
subsequent oxidation (Scheme 3). In addition to the electronic
factors responsible for the lowered oxidation potential, a
synergistic interplay between the π-conjugation and the
Figure 7. Oxidative cyclization of 8 (10 μM) to 14, triggered by
copper(II) ions (100 equiv) in water (pH = 7.0; HEPES, 50 mM) and
monitored by (a) UV−vis and (b) fluorescence spectroscopy after 10
min. Digital images in the insets were obtained (with ambient light for
(a), and a hand-held UV lamp (λexc = 365 nm) for (b)) for the samples
prepared using the same experimental conditions.
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by free copper(II) ion was observed, presumably due to the
weak metal-binding affinity of the triazole or N,N-diethanol-
amine groups in aqueous environment.
The practical utility of 8 was demonstrated further by the (i)
consistent turn-on fluorescence response to copper(II) in water
in the physiologically/environmentally relevant pH window of
6−8 (Figure 8a) but no fluorescence signaling at pH < 5.5,29
(ii) high sensitivity to the ppm-level concentrations of
copper(II) (Figure 8b), and (iii) high selectivity toward
copper(II) in the presence of various metal ions (Figure
8c,d).30 A plot of ΔI530 nm vs copper concentration in Figure 8b
shows an excellent linear relationship down to the value of
[Cu2+] = 10 μM, which is comparable to the U.S. Environ-
mental Protection Agency (EPA) guideline of 1.3 ppm (= 21
μM) copper(II) ion in drinking water.31
SUMMARY AND OUTLOOK
■
An efficient chemical transformation of nonemissive azoanilines
to highly fluorescent benzotriazoles was exploited for the turn-
on fluorescence detection of copper(II) ion in aqueous
solutions at room temperature. The most optimized system
responds selectively to ppm-level copper(II) at pH = 6−8 but
remains latent in acidic (pH < 5.5) environment. Comparative
kinetic and electrochemical studies on a series of model
compounds have established that the oxidation potential of the
precursor correlates directly with the rate of cyclization, which
is accelerated by an increasing number of amine substituents
installed on the common azoaniline platform. X-ray crystallo-
graphic and computational studies provided significant insights
into the potential functional role of intramolecular hydrogen
bonds and π-conjugated electron-donor groups in this
reactivity-based metal detection scheme.
In addition to the turn-on fluorescence signaling with a large
Stokes shift, the energy of the CT-type emission of the
cyclization product is strongly dependent on the solvent
polarity. Such features should be advantageous in tracking
copper(II) ions in biological samples, for which information on
the local dielectric could also be obtained in a simultaneous
fashion by polarity-sensitive shifts in the emission wavelength.
Efforts are currently underway in our laboratory to improve the
photophysical properties of this first-generation proof-of-
principle system for such advanced applications.
EXPERIMENTAL SECTION
■
General Considerations. All reagents were obtained from
commercial suppliers and used as received unless otherwise noted.
The solvents acetonitrile, hexane, tetrahydrofuran, and dichloro-
methane were saturated with nitrogen and purified by passage through
activated Al2O3 columns under nitrogen (Innovative Technology SPS
400). All air-sensitive manipulations were carried out under nitrogen
atmosphere by standard Schlenk-line techniques. The compounds
N,N-bis(2-hydroxyethyl)-4-amino-aniline32 and 4-tert-butyl nitroso-
benzene33 were prepared according to literature procedures.
Figure 8. (a) Changes in the emission spectra of 8 (10 μM) after
exposure to copper(II) (1.0 mM) in water at pH = 6.0−8.0 (MES
buffer for pH = 6.0−6.5; HEPES buffer for pH = 7.0−8.0; 50 mM).
(b) A plot of fluorescence intensity at λ = 530 nm of 8 (10 μM) vs
concentration of copper(II) in water at pH = 7.0 (HEPES, 50 mM).
(c) Fluorescence response of 8 (10 μM) toward metal ions in water at
pH = 7.0 (HEPES, 50 mM). The signal intensity at λ = 530 nm (= I)
is normalized with that of the probe-only sample (= I0). The bars
represent I/I0 of 8 in the presence of various metal ions (100 equiv)
screened. (d) Selectivity of 8 (10 μM) for copper(II) in the presence
of other metal ions. The light-gray bars represent the I/I0 value in the
presence of various metal ions (100 equiv) screened. The black bars
indicate the change in the emission intensity upon subsequent addition
of copper(II) (100 equiv) to the solution containing 8 and the metal
ion of interest. For all measurements, λexc = 380 nm; T = 298 K.
1
Physical Measurements. H NMR and 13C NMR spectra were
recorded on a 400 MHz Varian Inova NMR spectrometer. Chemical
shifts were reported versus tetramethylsilane and referenced to the
residual solvent peaks. High-resolution chemical ionization (CI)
(using CH4 as CI reagent) and electrospray ionization (ESI) mass
spectra were obtained on a Thermo Electron Corporation MAT
95XP-Trap. High-resolution GC-MS (CI, using CH4 as CI reagent)
was obtained on a Thermo Electron Corporation MAT 95XP-Trap.
FT-IR spectra were recorded on a Nicolet 510P FT-IR spectrometer
with EZ OMNIC ESP software. UV−vis spectra were recorded on an
Agilent 8453 UV−vis spectrophotometer with ChemStation. Fluo-
rescence spectra were recorded on a Photon Technology International
QM-4-CW spectrofluorometer with FeliX32 software.
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Fluorescence Quantum Yield Measurements. Quantum yields
were determined by standard methods,34 using coumarin 30 (ΦF =
0.67 in MeCN solution; λexc = 380 nm) or trans,trans-1,4-diphenyl-1,3-
butadiene (ΦF = 0.42 in hexane solution; λexc = 320 nm) as a standard.
The sample absorbance was maintained < 0.1 to minimize internal
absorption. Corrections were made to account for the differences in
solvent refractive indexes.
Reactivity Studies. A stock solution of 8 (5.0 mM) in DMSO was
diluted with buffered H2O (pH = 7.0; HEPES, 50 mM) to prepare
sample solutions (10 μM; 0.2% DMSO (v/v) in H2O). Solution
samples (40 mM) of metal ions Cu2+, Li+, Na+, K+, Ca2+, Mg2+, Mn2+,
Fe2+, Co2+, Ni2+, Zn2+, Cd2+, and Hg2+ were prepared by dissolving the
corresponding salt in water. In order to minimize dilution effects, a 75
μL aliquot of each metal ion stock solution (= 100 equiv with respect
to 8) was delivered using a microsyringe into a 3.0 mL sample solution
of 8 placed in a thermostatted Peltier cuvette holder.
Kinetic Studies. Comparative kinetic studies on 4−7 were carried
out in MeCN by delivering an aqueous solution sample (30−90 μL) of
Cu(OAc)2 into a MeCN solution sample (3.0 mL) of each reactant
placed in a thermostatted (T = 298 K) Peltier cuvette holder. The total
amount of water in this mixed solvent system was maintained constant
(90 μL; 3% H2O (v/v) in MeCN) by adding an appropriate (0−60
μL) amount of H2O to the solution prior to the injection of the
copper(II) reagent. With constant stirring, the time-dependent
changes in the fluorescence intensity were monitored at the maximum
emission wavelength of each compound: λ = 450 nm for 4 (λexc = 350
nm) and 6 (λexc = 360 nm); λ = 480 nm for 5 (λexc = 370 nm) and 7
(λexc = 380 nm). The ΔI vs t kinetic traces were fitted by a nonlinear
regression method (OriginPro 8.6) using eq 1, in which the
parameters k′ (= k[Cu2+]0) and I (= intensity at t → ∞) were
allowed to vary.
(114 mg, 0.364 mmol, yield = 92%). 1H NMR (400 MHz, DMSO-d6,
298 K): δ 7.95 (d, J = 9.3 Hz, 2H), 7.63 (d, J = 9.0 Hz, 1H), 6.89−6.92
(dd, J = 9.0, 2.0 Hz, 1H), 6.82 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 1.1 Hz,
1H), 5.48 (br s, 2H), 4.79 (br t, J = 5.5 Hz, 2H), 3.56−3.61 (q, J = 5.9
Hz, 4H), 3.47−3.50 (t, J = 5.8 Hz, 4H); 13C NMR (100 MHz, DMSO-
d6, 298 K): δ 148.4, 148.1, 146.5, 139.3, 129.4, 121.6, 121.0, 118.2,
111.8, 93.3, 58.5, 53.7. FT-IR (thin film on NaCl, cm−1): 3338, 3227,
2930, 2871, 1631, 1607, 1560, 1517, 1457, 1417, 1356, 1300, 1218,
1049, 1004, 816. HRMS (ESI) calcd for C16H20N5O2 [M + H]+
314.1617; found 314.1609.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and characterization of 4−7 and 10−13; additional
spectroscopic, kinetic, and X-ray crystallographic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
dongwhan@indiana.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by DTRA/ARO (W911NF-07-1-
0533).
Electrochemistry. Electrochemical studies were carried out under
ambient atmosphere with an Autolab Model PGSTA30 potentiostat
(Eco Chemie). A three-electrode configuration consisting of a working
electrode (glassy carbon electrode), a Ag/AgNO3 (0.01 M in MeCN
with 0.1 M nBu4NPF6) reference electrode, and a platinum coil
counter electrode was used. All electrochemical potentials are reported
to the Cp2Fe/Cp2Fe+ redox couple.
4-[((N,N-Bis(2-hydroxyethyl)-4-amino)phenyl)azo]-1,3-ben-
zenediamine (8). To a stirred MeOH solution (10 mL) of N,N-
bis(2-hydroxyethyl)-4-amino-aniline (1.73 g, 7.67 mmol) was added
slowly conc. HCl (2.5 mL). The reaction mixture was kept at 0 °C
using an ice bath. An aqueous solution (1 mL) of NaNO2 (0.63 g, 9.2
mmol) was added dropwise to generate the azonium intermediate, and
the reaction mixture was stirred for 10 min. A solution of m-
phenylenediamine (1.00 g, 9.27 mmol) and sodium hydroxide (1.00 g)
in MeOH−H2O (2:1, v/v; 15 mL) was kept at 0 °C. With stirring, the
azonium intermediate was added dropwise to the m-phenylenediamine
solution while maintaining the temperature of the reaction at 3−5 °C.
After stirring for 30 min, water (200 mL) was added to induce
precipitation of a red solid, which was isolated by filtration and washed
thoroughly with water to furnish 8 (2.26 g, 7.17 mmol, yield = 93%).
1H NMR (400 MHz, DMSO-d6, 298 K): δ 7.56 (d, J = 9.0 Hz, 2H),
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