Photophysical study of a polyoxo ethylene linked naphthalene-based fluorescent chemosensor for Mg2+ and Ca2+ detection

Article abstract of DOI:10.1039/c3pp00007a

A naphthalene-based bichromophoric fluorescent sensor 2,2′-[oxy- bis(2-oxatetramethyleneoxy)]-bis[N-(2-naphthyl)-benzamide)] (1) was synthesized and characterized. Fluorescence decay for 1 in alcoholic solvents in the region of 415-460 nm revealed bi-expo

Full text of DOI:10.1039/c3pp00007a

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Photophysical study of a polyoxo ethylene linked
naphthalene-based fluorescent chemosensor for Mg2+ and Ca2+
detection
Xiaoyan Xiang , Dan Wang，Yali Guo, Weisheng Liu , Wenwu Qin1
[Revised- Mar 27, 2013]
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu
Province and State Key Laboratory of Applied Organic Chemistry, College of
Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R.
China.
Abstract
A
naphthalene-based
bichromophoric
fluorescent
sensor
2,2´-[oxy-bis(2-oxatetramethyleneoxy)]-bis[N-(2-naphthyl)-benzamide)] (1) was
synthesized and characterized. Fluorescence decay for 1 in alcoholic solvents in the
region of 415-460 nm revealed bi-exponential behavior. The faster component of the
decay can be attributed to the formation of dimmers. Above 480 nm besides the dimer
there is also a little excimer formation and this excimer emits at longer wavelengths
than the dimer. The observation of the change of the fluorescence emission spectra
upon addition of water in EtOH-water mixtures is in line with formation of
water-bridged complexes preventing the excimer formation. The sensor shows an
increase of the fluorescence intensity upon increasing Mg2+ or Ca2+concentration in
EtOH because the formation of excimer can be hindered upon complexation with
Mg2+ or Ca2+ ions. Because of the competition between hydrated metal ion and
1
To whom correspondence should be addressed. E-mail for W. Qin: qinww@lzu.edu.cn, Tel.: +86
-931-8912582; Fax: +86-931-8912582
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water-bridged complex, spectral changes by complex with Mg2+ or Ca2+ in EtOH/H2O
(9/1 V/V) are quite different from those in neat ethanol. The ground-state dissociation
constants Kd estimated for the complex with Mg2+ or Ca2+was found to be around 2.0
mM in EtOH/H2O (9/1 V/V), which makes it suitable for the measurement of the
concentrations of these ions in physiologically relevant concentration ranges.
1. Introduction
Fluorescence spectroscopy is an attractive analytical technique to measure
intracellular concentrations of biologically important ions (Na+, K+, Mg2+, Ca2+, and
Cu2+) and molecules. Intracellular loading of a fluorescent probe leaves the cell intact
and thereby allows one to study the functioning cell.1,2,3 In almost all cells cytosolic
Mg2+ or Ca2+ is a crucial intracellular messenger regulating many cellular processes.
So it is important to develop new indicators for intracellular Mg2+ or Ca2+ with better
properties.
The choice of Mg2+ or Ca2+ indicators depends on the Mg2+ or Ca2+
concentration one intends to the measure. The ground-state dissociation constant (Kd)
of the formed Mg2+ or Ca2+ complex must be well-matched with the Mg2+ or Ca2+
concentration range of interest, because indicators have a usable fluorescence
response in the range from approximately 0.1 Kd to 10 Kd. Intracellular Mg2+ and Ca2+
concentrations can be quite different. For example, Mg2+ concentrations commonly
found in cells, typically range from about 0.1 mM to 6 mM.4,5 Intracellular free Mg2+
levels have been reported to be ~0.3 mM in synaptosomes,5 whereas the concentration
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of Mg2+ in normal serum is ~0.44–1.5 mM.6,7
Crown ethers are well-known to bind alkali and alkaline-earth metal ions8,9 in the
form of rigid and highly stable complexes.10 The corresponding polyoxo ethylene
chain (or open-chain crown ether or acyclic polyether) shows only weak binding, but
the conformation usually can be changed from a linear to a pseudocyclic structure
upon complex formation with metal ions. When this conformational change could be
converted into detectable signal, such as absorption and fluorescence, it would be
possible to develop them as metal ion sensors. Early studies on polyoxo ethylene
chain derivatives were carried out by Vögtle et al.11,12 After that, polyoxo ethylene
chains -based sensors have been developed. They achieve the signal of the binding
event mainly by excimer formation, 13 twisted intermolecular charge transfer
(TICT)14,15 process, and PET16 process between two terminal chromophore units.
The so-called excimer refers to a complex that is formed by two identical
fluorophores in the excited state, and which is not stable in the ground state.
Fluorescence properties will be changed when excimer can be formed. 17 , 18
Nakamura’s group synthesized new fluorescent probes that have two fluorescent
chromophores (naphthalene anthracene, pyrene, fluorene, etc.) symmetrically or
asymmetrically placed at both terminals of 1,13-diamino-4,7,10-trioxatridecane, and
examined their complexations with alkali and alkaline earth cations.19,20 In the
addition of alkali and alkaline earth metal ions, there is an enhancement of monomer
emission peak together with a weakening of the excimer emission peak.
In a previous study, we synthesized a polyoxo ethylene chain alkaline earth
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metal fluorescent sensor that contained 2-aminoanthracene terminal chromophores.21
Its ground-state dissociation constant Kd for Ca2+ is about 0.1 mM. This sensor can be
considered as low-affinity Ca2+ indicator − with Ca2+ dissociation constants above 1
µM. Other low-affinity Ca2+ probes, such as mag-fura-2, mag-fura-5 and mag-indo-1,
were originally designed to report intracellular Mg2+ levels (sensitive to Mg2+
concentrations from 0.1 to 6 mM). Typical physiological Ca2+ concentrations (10 nM
– 1 µM) do not usually interfere with Mg2+ changes because of the relatively low
affinity of these indicators. So this sensor shows potential for sensing for Mg 2+ levels
in physiological condition. But the photophysical properties of this bichromophoric
sensor containing 2-aminoanthracene is quite complex, because it can form
intramolecular excimers and intermolecular excimers in parallel. We also investigated
22
photophysical properties of a series of adamantanebisurea derivatives.
Fluorescence decay for both compounds in 1-naphthyl derivatives revealed
bi-exponential behavior. On the contrary, for 2-aminoanthracene and 1-aminopyrene
derivatives the decay becomes three-exponential due to the biggest π-surface for the
π,π-stacking. This finding means that the photophysical properties of anthracene or
pyrene derivatives are quite more complex than those of naphthyl derivatives.
Based on the results of above research, in the present paper a new fluorescent
probe 1 based on polyoxo ethylene chain derivatives with 1-naphthalene as terminal
group is presented. The solvent-dependent photophysical characteristics of probe 1
and complex formation of 1 with Mg2+ or Ca2+ ions was spectroscopically studied.
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2. Experimental Section
2.1. Instruments and reagents
13
1H and C NMR spectra were taken on a Bruker DRX-400 spectrometer with
TMS as an internal standard and DMSO as solvent. Mass spectra were recorded on a
Bruker Daltonics esquire6000 Mass spectrometer. Chemical shift multiplicities are
13
reported as s = singlet, d = doublet, t = triplet and m = multiplet. C spectra were
referenced to the DMSO (39.70 ppm) signal. Mass spectra were recorded in E.I. mode.
Melting points were taken on an X-4 precise micro melting point cryoscope (Beijing
Fukai Instrument Co.) and are uncorrected. N-Acetyl-1-aminonaphthalene was
purchased from TCI. All the materials for synthesis were purchased from commercial
suppliers and used without further purification. Deionized water was used as solvents.
Perchlorate salts of magnesium, calcium were of spectroscopic grade and used as
received. The solutions of other metal ions were prepared from their perchlorate salts.
2.2. Syntheses
2,2´-[oxy-bi(2-oxatetramethyleneoxy)]-bi[N-(2-naphthyl)-benzamide)] (1)
The synthetic pathway of compound 1 is shown in Scheme 1. Compound 2 was
synthesized following a literature procedure.23
Preparation of diacid dichlorides
The dicarboxylic acid 2 (0.025 mol) was heated in thionyl chloride (50 ml) for
°
12h at 50-60 C. The thionyl chloride was evaporated at low temperature to give the
diacid dichloride as a light yellow solid in 80﹪yield which was applied in the
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subsequent coupling step without any other purification.
General procedure for synthesis of diamide
0.31g (2.1 mmol)of 1-naphthylamine was dissolved in 80 mL of acetonitrile, and
2.0 mL of triethylamine. The mixture was cooled in an ice bath and a solution of the
above diacid chloride in 30 mL of acetonitrile was added dropwise. The mixture was
stirred for 24 h at room temperature. The solvent was evaporated under reduced
pressure, and the residue was dissolved in 30 mL chloroform, then washed with 20 ml
of water three times, and dried over MgSO4. The crude product was purified by silica
gel column chromatography (eluent: chloroform-ethyl acetate-petroleum). Yield: 31
°
1
%. mp 121-123 C. H NMR(DMSO-d6, 400 MHz): δ=2.97(−C−CH2−O, Ha, 4H),
3.51(−C−CH2−O, Hb, 4H), 4.15 (−C−CH2−O, Hc, 4H), 7.16 (aromatic, m, 4H),
7.49-7.54(aromatic, m, 8H), 7.76 (aromatic, d, 2H, J=8.0 Hz), 7.87 (aromatic, d, 2H,
J=9.2 Hz), 7.93 (aromatic, d, 2H, J=4.8 Hz), 7.99 (aromatic, d, 4H, J=8.0 Hz), 10.28
(NH, s, 2H); 13C NMR (DMSO-d6, 100 MHz,): δ68.65(t, 2C), 69.19(s, 2C), 79.16(s,
2C), 113.65(s, 2C), 115.73(s, 2C), 121.48 (t, 2C), 122.22 (t, 2C), 124.86(s, 2C),
125.53 (2C), 126.00(s,2C), 127.60 (d, 2C), 128.16(s, 2C), 130.00(s, 2C), 131.05(s,
2C), 132.50 (d, 2C), 133.34 (t, 2C), 154.95(s, 2C), 156.21(s, 2C), 162.73(s,2C),
163.84 (s, 2C). IR (KBr) νmax/cm-1 924-416(m), 1039(s), 1117(s), 1221 (s), 1269 (s),
1344 (m), 1398 (s), 1478 (s), 1500 (m), 1539 (s), 1598 (s), 1664 (s), 1722(m), 2854
(s), 2924 (s), 3371 (s), 3433 (s); MS (ESI ) m/z calcd. For C40H36N2O6 640.3; found
641.4(M+1, 100%).
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NH2
a
b
c
O
O
O
2
O
O
O
2
O C
NH
C O
NH
HOOC
COOH
i)SOCl2
ii)NEt3
2
1
Scheme 1. Synthesis of compound 1.
2.3. Steady-state UV–vis absorption and fluorescence spectroscopy
UV–vis absorption spectra were recorded on a Varian UV-Cary100
spectrophotometer. For the corrected steady-state excitation and emission spectra, a
Hitachi F-4500 spectrofluorometer was employed. Freshly prepared samples in 1-cm
quartz cells were used to perform all UV–vis absorption and emission measurements.
For the determination of the fluorescence quantum yields of 1, only dilute solutions
with an absorbance below 0.1 at the excitation wavelength (λex = 310 nm) were used.
For the measurement of fluorescence emission spectra as the function of
concentration of 1, some of the absorbance is higher than 0.1. 9-Cyanoanthracene in
MeOH (Φf = 0.80) was used as fluorescence standard.24 The majority of the Φf
determinations were done using non-degassed samples. In all cases, correction for the
solvent refractive index was applied. All spectra were recorded at 20 °C using
non-degassed samples. To check the influence of dissolved O2 on Φf, some samples
were purged with nitrogen for 15 minutes prior to measurement. The obtained Φf
values within experimental error were equal for aerated and degassed samples. The
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titration experiments with Mg2+ or Ca2+ metal were carried out by adding small
quantities of a stock solution of alkaline earth metal perchlorate salts in ethanol to a
much larger volume (25 ml) of solutions of 1.
2.4. Time-Resolved Fluorescence Spectroscopy
Fluorescence lifetimes were measured by means of an Edinburgh Instruments
FLS920 equipped with a light emitting diode (excitation wavelength 300 nm) and a
hydrogen flash lamp, using the time-correlated single photon counting technique in
2048 channels at room temperature. The samples were dissolved in ethanol and the
concentrations were adjusted to have optical densities at the excitation wavelength
(310 or 300 nm) ≈ 0.1. The monitored wavelengths were 415 nm, 430 nm, 460 nm,
480 nm, 500 nm and 520 nm.
Histograms of the instrument response functions (using a LUDOX scatter) and
sample decays were recorded until they typically reached 5.0×103 counts in the peak
channel. Obtained histograms were fitted as sums of the exponential, using
Gaussian-weighted nonlinear least squares fitting based on Marquardt-Levenberg
minimization implemented in the software package of the instrument. The fitting
parameters (decay times and preexponential factors) were determined by minimizing
the reduced chi-square χ2. An additional graphical method was used to judge the
quality of the fit that included plots of surfaces (“carpets”) of the weighted residuals
vs channel number. All curve fittings presented here had χ2 values < 1.1. A global
fitting of fluorescence decays was done by using FAST software supplied by
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Edinburgh Instruments Company. The global (linkable) fitting parameters are τ1 and
τ2. All global curve fittings presented here had χ2 values < 1.1.
g
2.5. Determination of Kd via direct fluorescence titration
The ground-state dissociation constants Kd of the complexes between 1 and
various ions were determined in EtOH/H2O (9/1 V/V) at 20 °C by direct fluorescence
titration as a function of the ion concentration using the emission spectra. Nonlinear
25
fitting of eq (1)
to the steady-state fluorescence data F recorded as a function of
[Mg2+] or [Ca2+] yields values of Kd, Fmin, Fmax, and n.
n
Fmax
[
M
]
+ Fmin Kd
[ ]
M
F =
(1)
n
Kd +
In eq 1, F stands for the fluorescence signal at [Mg2+] or [Ca2+], whereas Fmin
and Fmax denote the fluorescence signals at minimal and maximal [Mg2+] or [Ca2+],
respectively, and n is the number of ions bound per probe molecule (i.e.,
stoichiometry of binding). Because the fits of eq 1 to the fluorescence data F with n,
Kd, Fmin, and Fmax as freely adjustable parameters always gave values of n close to 1
(indicative of a 1:1 complex), n was kept fixed at 1 in the final curve fittings, from
which the estimated Kd values are reported here.
3. Results and discussion
3.1. Spectroscopic properties of 1 in alcoholic solvents
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Figure 1. (a) Normalized absorption spectra of 1 in several solvents. (b)
Corresponding normalized fluorescence emission spectra (λex = 310 nm).
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Figure S1 show the normalized UV/vis absorption and fluorescence spectra of
N-Acetyl-1-aminonaphthalene in ethanol and 1-pentanol. The absorption spectra has a
main band of similar shape as that of 1-aminonaphthalene (~ 310 nm) in other
solvents shown in the literature,26 with an absorption maximum at ∼300 nm. It has
two fluorescence emission peaks (centered at about 350 and 400 nm, respectively, Fig.
S1b).
UV/vis absorption and fluorescence emission spectra of 1 in different alcoholic
solvents are depicted in Figure 1. The absorption spectra of 1 show an intense
absorption band with a maximum λabs(max) located around 310 nm in all solvents
studied (Figure 1a). The absorption spectra are of similar shape as those of
N-Acetyl-1-aminonaphthalene. Compound 1 in ethanol shows an emission spectrum
with a maximum at ~437 nm. The maximum wavelength of the fluorescence band is
slightly red-shifted when the solvent is changed from ethanol (437 nm) to pentanol
(439 nm) or octanol (440 nm) (Figure 1b). The total fluorescence quantum yields are
0.08 in ethanol vs 0.37 in 1-pentanol，and 0.42 in 1-octanol.
To investigate the fluorescence dynamics of N-Acetyl-1-aminonaphthalene and 1,
fluorescence decay traces in different alcoholic solvents were collected as a function
of emission wavelength λem. The results of the time-resolved fluorescence
experiments are shown in Table 1, Table S1 and Figure S2.
The fluorescence decay of N-Acetyl-1-aminonaphthalene is fitted to the bi-
exponential profile with the lifetime of 5.9 ns and 1.5 ns in ethanol in the region of
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415-460 nm. In 1-pentanol, the fluorescence decays also show two main components,
with time constants of about 8.6 and 1.8 ns (Figure S2, Table S1). The contribution
from the τ1 component decreases, whereas that of the τ2 component increases with
increasing emission wavelength in both solvents. In additions, contributions of the
faster decay time τ2 increases with increasing the concentration of
N-Acetyl-1-aminonaphthalene in 1-pentanol (Table S1). Biexponential decay of
1-aminonaphthalenes has been reported.27 It may be attributed to the population of a
CT state in the polar solvent,28 the presence of La and Lb excited states of the
1-aminonaphthalene chromophore,29 to the presence of rotamers with different
singlet excited state lifetimes, or intermolecular or intermolecular excimer or exciplex
states. N-Acetyl-1-aminonaphthalene can in principle form dimmer or aggregate due
to the π,π-stacking of the aromatic rings.30,31 If one assumes that 10 % of the excited
monomers undergo excimer formation the pseudomonomolecular rate constant for
excimer formation would be ~1.7x107 s-1 based on the monomer decay time 5.9 ns in
ethanol, at a concentration of 10 µM this would correspond to a bimolecular rate
constant of 1.7x1012 M-1s-1. This is about 100 times larger than the rate constant for a
diffusion controlled process in the solvents used. So attributing the bi- exponential to
excimer formation is highly unlikely. Most probably there already had equilibrium
between monomers and dimmers in the ground state. Then slow decay might be
attributed to the lifetime of monomers molecules，while fast decay maybe could be
attributed to the lifetime of dimers.
Fluorescence decay for 1 in all solvents also revealed bi-exponential behavior in
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the region of 415-460 nm. The fluorescence decay is fitted to the bi-exponential
profile with lifetimes of 5.5 ns and 1.2 ns in ethanol. And the contributions from the
fast decay increased at longer wavelengths (from 56 % at 415 nm to 64 % at 460 nm)
(Figure 2a). The results of fluorescence decay of 1 are similar to those of
N-Acetyl-1-aminonaphthalene.
Figure S3 shown the normalized fluorescence emission spectra of 1 as the
function of concentration (6.3-10.0 µM) in ethanol. When the fluorescence emission
spectra are normalized at the maximum at 437 nm, then the blue side (350 -400 nm)
of the band is much less prominent at the higher concentrations. On the other side,
when the fluorescence emission spectra are normalized at the blue side (360 nm), then
the fluorescence intensity of red edge of the emission (400-550 nm) spectrum increase
with increasing concentrations.
The time-resolved fluorescence profiles of 1 in ethanol were measured as a
function of emission wavelength (λem = 415, 430, and 460 nm) and analyzed globally
as bi-exponentials with linked decay times τ1 (slow) and τ2 (fast). Measurements of
fluorescence decay traces as a function of λem in the region of 415-460 nm revealed a
decrease of the relative amplitude associated with the slow component (τ1) with
increasing detection wavelength λem (Table 1). The relative amplitudes α1 (%)
decreases with increasing λem (α1 =44 % at 415nm, α1 =38 % at 430nm, α1 =36 % at
460nm).
The major decay component with τ1=~5.5 ns in ethanol could be attributed to the
lifetime of emission of the monomer of 1, while ~1.2 ns could be attributed to the
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lifetime of dimer.
Above 460 nm, at 480, 500 and 520 nm, the emission is triple-exponential, a fast
rise of the emission τ3=0.23 ns could be detected. For compound 1 intramolecular
excimer formation cannot be excluded completely a priori as the structure. Negative
values in the relative amplitudes α3 (%) indicate rising up of excimer from monomer
(Table 1 and Figure S10). This fastest decay could be due to intramolecular excimer.
A slow decay component with a decay time τ1 =3.7 ns is smaller than 5.5 ns. Similar
photophysical properties for the fluorescent reagents that bearing two pyrenes
connected via an amide linkage at the ortho position of the benzene ring at the end of
the polyether (~150 ns at 400 nm due to monomer emissions of pyrene becoming ~45
ns at 480 nm) have been reported before.13 This means that besides the dimer there is
also a little excimer formation and this excimer emits at longer wavelengths than the
dimer.
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Table 1. Global analyst results of sensor 1 (10 µM) in alcoholic solvents, and in
addition of Mg2+ (6.0 mM) or Ca2+ (7.5 mM) in ethanol. Decay times τ1, τ2 and τ3, and
the relative amplitude (%).
compound
solvent
monitored
wavelength/
nm
τ1/ns
τ2/ns
τ3/ns
α1
α2
α3
(%)
(%) (%)
415
430
460
480
500
520
44% 56%
38% 62%
36% 64%
5.50±0.001 1.19±0.005
ethanol
6% 88% -6%
4% 74% -22%
4% 75% -21%
3.74±0.006 0.89±0.007 0.23±0.20
7.33±0.001 1.90±0.007
1
415
430
460
415
430
460
415
430
80% 20%
77% 23%
63% 37%
79% 21%
65% 35%
50% 50%
83% 17%
71% 29%
1-pentanol
1-Octanol
ethanol
6.79±0.001 1.97±0.006
6.24±0.001 0.81±0.024
1-Mg2+
1-Ca2+
ethanol
415
430
85% 15%
63%) 37%
6.25±0.001 1.20±0.029
Fluorescence decay of 1 in 1-pentanol and 1-octanol solvents also revealed
bi-exponential behavior in the region of 415-460 nm, but the slow decay becomes
longer and has more contribution. This means that there are more monomer and less
dimer in a more viscous solvent. In line with the fluorescence quantum yield of 1 in
both solvents is higher than that in ethanol.
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Figure 2. The decay curves of 1 collected at different wavelengths in (a) ethanol and
(b) 1-pentanol.
3.2. Complex formation between 1 and Mg2+ or Ca2+ metal ions in neat ethanol
solvent
The formation of intramolecular excimer can be influenced by complex
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formation with metal ions. This will cause the change of the fluorescence
spectroscopic properties. Complex formation was investigated for 1 with alkali (Li+,
Na+ and K+), alkaline-earth (Mg2+, Ca2+ and Ba2+), and transition metal ions (Al3+,
Ni2+, Mn2+,Cu2+, Zn2+, Cd2+, Co2+, Cr3+, Fe3+and Hg2+) in EtOH.
Upon addition of the alkali ions Li+, Na+ and K+ and the alkaline-earth ion Ba2+
and transition metal ions to a solution of 1 in EtOH, no change in the UV–vis
absorption and fluorescence spectra could be found.
Conversely, the alkaline-earth ions Mg2+ and Ca2+ produced spectral changes of
1. The absorption band of 1 slightly decreases when Mg2+ ions are added into the
EtOH. In salt-free ethanol, the emission spectrum of 1 displays one peak with the
maximum at 437 nm (figure 3a) with fluorescence quantum yield of 0.08. The low Φf
value for free 1 in ethanol can be attributed to the formation of dimmer and excimer,
which has a lower fluorescence quantum yield than the excited “monomer”. However,
the maximum of the fluorescence emission band shifts from 437 nm to 410 nm in
EtOH with fluorescence quantum yield is 0.34. This may be due to the ability of Mg2+
to break up the excimer by forming a different complex with 1. Moreover, the reaction
of a metal ion with a chelating agent induces rigidity in the resulting molecule and
tends to produce a large chelation enhanced fluorescenc (CHEF) effect which induces
the large enhancement of fluorescence. The highest fluorescence quantum yield value
(0.34 for excitation at 310 nm) is found at about 6.0 mM Mg2+.
From the analyses of all the spectra and fluorescence titrations of 1 with Mg2+
ions it is clear that a stoichiometry of 1:1 (ligand : cation) is found over the range 0.0
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mM - 6.0 mM. The Kd value for the Mg2+ complex with 1 was obtained from direct
fluorimetric titrations (eq 1) using emission spectra is Kd = 0.6 mM and the fitting is
in agreement with a well-defined 1:1 stoichiometry between 1 and Mg2+(Figure 3b).
Figure 3. (a) Fluorescence emission (λex = 310 nm) spectra of 1 (10 µM) in EtOH as a
probe of [Mg2+]. (b) Best fit of eq 1with n = 1 to the direct emission fluorescence
titration data of 1 obtained from the spectra of 3a (λex = 310 nm, λem = 410 nm).
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The emission spectral changes of 1 in the presence of Ca2+ (Figure S4a) are
similar to those of Mg2+. The φf value of 1 in the presence of Ca2+ is 0.23. The average
Kd value of the 1- Ca2+ complex estimated via direct fluorometric titrations using
excitation spectra was found to be 2.0 mM. From the analyses of all the spectra and
fluorescence titrations of 1 with Ca2+ ions it is clear that a stoichiometry of 1:1
(ligand:cation) is found with [Ca2+] over the 0.0 mM to 7.5 mM range（Figure S4b）.
The complex formation between 1 and Mg2+ or Ca2+was also investigated by
time-resolved fluorescence. Figure 4 presents the fluorescence decay of 1 before and
after addition of Mg2+ or Ca2+ in ethanol. Following addition of Mg2+ or Ca2+ to 1, the
longer lifetime increases (∼5.5 to ∼6.2 ns) along with an increase in the amplitude
(∼40% to ∼84%). The shorter component remains constant (∼1.0 ns), and in general,
the contribution from this component decreases (∼60% to ∼16%) (Table 1).
Figure 4. The decay curves of 1 (10 µM) before and after addition of Mg2+ or Ca2+
(6.0 mM) in ethanol collected at 415nm when excited at 310 nm.
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3.3 Effect of water on the emission spectra of 1
The addition of water does not induce significant changes in the absorption
spectrum of 1 in ethanol, suggesting only weak ground state interactions.
The fluorescence emission spectra, however, change considerably, as shown in
Figure 5. The maximum of the fluorescence emission band shifts from 437 nm in
ethanol to 407 nm upon the addition of water and is accompanied by an increase in
intensity. The likely reason is that an intermolecular hydrogen bond can be formed
between two amide groups with water as a bridge. The H2O···H2O hydrogen bonds
can easily form even at lower water content in water–EtOH mixed solution.
Observation of the change of the fluorescence emission spectra in addition of water is
in line with formation of water-bridged complex and preventing the dimmer or
excimer.
Mono-exponential decays with a lifetime of 6.56 ns in DMSO and 7.50 ns in
MeCN also have been found in the case of other compounds that only contain a single
1-amino-naphthalene chromophore comprised of a rigid adamantane moiety.22 This
suggests that aggregation or formation of the excimer is not possible due to the
sterical hindrance of the adamantane. The formation of water-bridged complex can
increase the fluorescence intensity of 1, this could be due to specific binding of water
between the amides, but also the PEG chain could be more extended when solvated
with more water, preventing the formation of dimmer or excimer which is formed by
intermolecular H-bonds between the NH moieties or π,π-stacking.
The effects of water on the emission spectra of two cupreidine derivatives in
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32
methanol–water mixed solutions were already reported by Brouwer et al.
Complexation with water leads to enhanced excited state proton transfer (ESPT) can
explain this change. In this experiment, proton transfer in the excited state maybe also
33
is possible. The N-H becomes a lot more acidic, just like in naphthols,
complexation with water leads to enhanced excited state proton transfer
Figure 5. Emission (λex = 310 nm) of 1.0 × 10-5 M of 1 in ethanol containing various
concentrations of water (v/v). Inset is the fluorescence intensity variation trend vs.v/v
water at 400 nm.
.
3.4. Complex formation between 1 with Mg2+ or Ca2+ in EtOH/H2O (9/1 V/V)
Since water has a large effect on the fluorescence emission spectra of 1 in
ethanol, one may wonder if the fluorescence emission spectral changes by complex
with Mg2+ or Ca2+ in EtOH-H2O mixture the same as those in neat ethanol. To
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investigate this, spectra of 1 as a function of [Mg2+] or [Ca2+ ] were measured in
EtOH/H2O (9/1 V/V).
The addition of Mg2+ to a solution of 1 in EtOH/H2O (9/1 V/V) affects the
absorption spectra. Upon increasing the metal ion concentration, the main absorption
band (with maximum at 310 nm in the absence of metal ions) decreases in intensity
and blue shifts to 300 nm (figure S5).
The fluorescence emission spectra measured as a function of Mg2+concentration
in EtOH/H2O (9/1 V/V) are shown in figure 6a. The fluorescence intensity at 407 nm
first decreased upon addition of 0.1 mM Mg2+ to a solution of 1. The intensity
decrease to extent as 1 in neat ethanol, with fluorescence quantum yield is 0.08. With
further increasing the magnesium ion concentration, fluorescence intensity increases
gradually. When a small quantity of magnesium (0.1 mM Mg2+) is present, the
formation hydrated magnesium ion can break water-bridged complex, which will
decrease the fluorescence intensity. Above this range, the fluorescence emission
increases its intensity with further increasing the magnesium ion concentration by
complex formation with magnesium ions. The highest fluorescence quantum yield
value (0.45 for excitation at 310 nm) is found about 3.3 mM Mg2+ in EtOH/H2O (9/1
V/V) solution.
The Kd value for the Mg2+ complex with 1 was obtained from direct fluorimetric
titrations (eq 1) using emission spectra is 2.0 mM and gave a well-defined 1:1
stoichiometry between 1 and Mg2+ ( from 0.10 mM to 3.33 mM)(Figure 6b).
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Figure 6. (a) Fluorescence emission (λex = 310 nm) spectra of 1 (10 µM) in
EtOH/H2O (9/1 V/V) as a probe of [Mg2+]. Inset is the fluorescence intensity variation
trend as the function of [Mg2+]. (b) Best fit of eq 1 with n = 1 to the direct emission
fluorescence titration data of 1 obtained from the spectra of Fig 6a (from 0.10 mM to
3.33 mM) (λex = 310 nm, λem = 407 nm).
The fluorescence spectral changes has been observed when Ca2+ was added into
the EtOH/H2O (9/1 V/V) solution of 1 are very similar to those for Mg2+. But the
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enhancement is smaller than that of Mg2+. The highest fluorescence quantum yield
value (0.25 for excitation at 310 nm) is found above 1.23 mM Ca2+ in EtOH/H2O (9/1
V/V) solution. A Kd value of 2.0 mM was obtained for 1:1 complex with Ca2+ (from
0.05 mM to 1.23 mM) by direct fluorimetric titration (Figure S6), which is the same
as in neat ethanol. Schematic 2 showed the representation of the formation of
water-bridged complex of 1 before and after addition of Mg2+or Ca2+ in EtOH/H2O
(9/1 V/V).
O
O
O
O
O
O
O
O
O
O
N
H2O
O
O
C
C
H
C
N H
C
N H
O
HN
H
H
Water-bridged complex
(φ=0.21)
(φ=0.08)
1
0.10mM Mg2+
or
0.05mM Ca2+
M=Mg or Ca
O
M2+
O
O
O
C
Mg2+ or Ca2+
O
C
O
O
O
H
O
O
O
O
H
O
C
C
N H
H
M2+
HN
H
HN
H
N H
H
O
O
H
H
O
M2+
H
H
O
M2+
H
M2+
O
O
O
H
H
H
H
H
M2+ complex with 1
(M=Mg, φ=0.45)
(M=Ca, φ=0.25)
Hydronium metal ion
(φ=0.08)
Scheme 2. Representation of the structures of 1 before and after addition of Mg2+or
Ca2+ in EtOH/H2O (9/1 V/V).
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A near-linear correlation between fluorescence intensity and concentration of
Mg2+ (r = 0.992, n ≥ 9 ) was obtained over the range 0.1–1.0 mM Mg2+. The detection
limit was 1.0 µM, which was calculated by multiplying the standard derivation of 10
blank measurements by 3 and dividing by the slope of the linear calibration) of sensor
1 between intensity of fluorescence emission at 407 nm vs magnesium ion
concentration in EtOH/H2O (9/1 V/V) was obtained. Similarly, a near-linear
correlation(r = 0.992, n ≥ 12) was obtained over the range 0.05–1.08 mM Ca2+ (Figure
S8). This demonstrates the potential utility of sensor 1 for calibrating and determining
magnesium or calcium ion concentration in aqueous solutions.
It is known that achieving a high selectivity for the analyte of interest over a
complex background of potentially competing species is a challenging task in sensor
development. Figure 7 shows the fluorescence response of 1 to Mg2+ in the presence
of 10 times competitive ions in EtOH/H2O (9/1 V/V). Except for Fe3+, a background
of most selected coexistent metal ions does not interfere with the sensing of 1 for
Mg2+. However, the addition of 1 equiv of masking agent NaF to the solution of 1
containing Fe3+ and Mg2+ in EtOH/H2O (9/1 V/V) weakens the influence of Fe3+.34 In
addtion, since actually physiological Fe3+ concentrations is no so high, then they do
not interfere with Mg2+ in living cells. Figure S9 shows the fluorescence response of 1
to Ca2+ in the presence of 10 times competitive ions in EtOH/H2O (9/1 V/V).
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Figure 7. Selectivity of 1 (10 µM) for Mg2+ (3.0 mM) over other cations. The bars
indicate the fluorescence change that occurs immediately following the addition of
interfering ions (10 equiv. of the Mg2+ ions) to the EtOH/H2O (9/1 V/V)solution. The
emission wavelength is 407 nm.
The main infrared bands of the sensor and its complexes are presented in Figure
8. The IR spectra of Mg2+ and Ca2+ perchlorate of 1 are relatively similar, but not the
same as the infrared absorption of the 1 suggesting that 1 and the complexes have
similar coordination modes. As is shown in the IR spectra of 1 (1664 cm-1), the IR
spectra of carbonyl absorption peaks of (C = O) of the complex (1639 cm-1-1643 cm-1)
have red shift of about 20 cm -1, indicating that the ligand in the carbonyl oxygen
atoms involved in the coordination of Mg2+ or Ca2+ ions .
When the ions bind to the polyether and to the carbonyls (as in Scheme 2),
because the amide groups are initially weakly electron donating, being bound via the
N-atom, but the ions reduce the electron density of the amide unit. So when Mg2+ or
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Ca2+ is bound to 1 in ethanol, the emission spectrum shifts to the blue.
Before the formation of the complex, 1 shows weak fluorescence emission due to
the dimmer and excimer formation by the two molecules through π-π stacke or
intermolecular H-bonds between the NH moieties. After being bound with Mg2+ or
Ca2+, the complex formed a rigid conformational structure. The reduction of the
conformational flexibility probably hinders dimmer and excimer formation, which
will results in a fluorescence emission intensity enhancement.
Figure 8. FTIR spectra for 1 and its Mg2+ or Ca2+ complexes (solid sate).
4. Conclusion
In this report, a polyoxo ethylene chain fluorescent sensor 1 with two
1-naphthylamine chromophores as terminal group was synthesized. Fluorescence
decay for 1 in alcoholic solvents in the region of 415-460 nm revealed bi-exponential
behavior whereas the faster decay can be attributed to dimmer. Above 480 nm besides
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the dimer there is also a little excimer formation and this excimer emits at longer
wavelengths than the dimer. The formation of water-bridged complex can break the
dimmer and eximer which will lead to increase fluorescence intensity and shifts the
spectra. But no UV/vis absorption spectra change can be found in addition of water.
Other similar polioxo ethylene chain probes have been studied before. But the study
of the binding properties of these probes with metal ions was only carried out in
organic solvents. In this work, the observation of the change of the fluorescence
emission spectra upon addition of water in EtOH-water mixtures is quite interesting
because samples to be studied are always aqueous in practice. The major purpose of
this work is to investigate the different photophysical properties of polioxo ethylene
chain sensor 1 between in EtOH/H2O (9/1 V/V) and in neat ethanol.
Complex with Mg2+ or Ca2+ will hider the formation of dimmer and eximer and
cause the fluorescence emission intensity increase. In addition, the UV/vis absorption
spectra also change as the function Mg2+ or Ca2+.
The competition between hydrated metal ion and water-bridged complex led the
fluorescence emission spectra change of complex with Mg2+ or Ca2+ in neat ethanol
and in EtOH/H2O (9/1 V/V) are different. The sensor forms 1:1 complexes with Mg2+
or Ca2+ with the ground-state dissociation constants Kd were found to be around 2 mM
in EtOH/H2O (9/1 V/V).
Supporting Information Available:
Compound characterization data, absorption, fluorescence emission spectra of 1 in
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ethanol as a probe of Mg2+ or Ca2+, this material can be found in the Supporting
Information.
Acknowledgement
We thank Prof. Fred Brouwer (University of Amsterdam) for helpful discussions.
This work was supported by the Chinese “Program for New Century Excellent
Talents in University” (NCET-09-0444), the ”Fundamental Research Funds for the
Central Universities” (lzujbky-2011-22 and lzujbky-2012-k13),
the National
Science Foundation for Fostering Talents in Basic Research of the National Natural
Science Foundation of China (Grant No. J1103307) and the “International
Cooperation Program of Gansu Province”(1104WCGA182). The authors would like
to thank the Natural Science Foundation of China (No. 21271094), and this study was
supported in part by the “Key Program of National Natural Science Foundation of
China” (20931003).
29