A new fluorescent sensor based on 1H-pyrazolo[3,4-b]quinoline skeleton. Part 2

Article abstract of DOI:10.1007/s10895-010-0726-x

A novel fluorescent dye bis-(pyridin-2-yl-methyl)-(1,3,4-triphenyl-1H- pyrazolo[3,4-b]quinolin-6-ylmethyl)-amine (P1) has been synthesized and investigated by means of steady state and time-resolved fluorescence techniques. This compound acts as sensor fo

Full text of DOI:10.1007/s10895-010-0726-x

J Fluoresc (2011) 21:375–383
DOI 10.1007/s10895-010-0726-x
ORIGINAL PAPER
A New Fluorescent Sensor Based
on 1H-pyrazolo[3,4-b]quinoline Skeleton. Part 2
Marek Mac & Tomasz Uchacz & Andrzej Danel &
Krzysztof Danel & Przemyslaw Kolek & Ewa Kulig
Received: 29 April 2010 /Accepted: 8 September 2010 /Published online: 2 October 2010
#
The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract A novel fluorescent dye bis-(pyridin-2-yl-methyl)-
(1,3,4-triphenyl-1H-pyrazolo[3,4-b]quinolin-6-ylmethyl)-
amine (P1) has been synthesized and investigated by means
of steady state and time-resolved fluorescence techniques.
This compound acts as sensor for fluorescence detection of
small inorganic cations (lithium, sodium, barium, magne-
sium, calcium, and zinc) in highly polar solvents such as
acetonitrile. The mechanism which allows application of this
compound as sensor is an electron transfer from the electron-
donative part of molecule (amine) to the acceptor part
(pyrazoloquinoline derivative), which is retarded upon
complexation of the electro-donative part by inorganic
cations. The binding constants are strongly dependent on
the charge density of the analyzed cations. The 2/1
complexes of P1 with Zn++ and Mg++ cations posses large
binding constants. Moreover, in the presence of these cations
a significant bathochromic shift of fluorescence is observed.
The most probable explanation of such behaviour is the
formation of intramolecular excimer. This is partially
supported by the quantum chemical calculations.
Introduction
Molecular systems based on 1,3,4-triphenyl1H-pyrazolo
[3,4-b]quinoline fluorophore were found to be good sensors
[1] for small inorganic cations also in less polar solvents as
tetrahydrofuran. Such compounds are attractive for fluores-
cence detection of nonfluorescent analytes such as metal
ions (sodium, lithium, calcium, barium, etc.) in biological
systems [2, 3], These cations are involved in many
biological processes of fundamental importance for life
and monitoring of these ions occurring in small amounts in
blood and urine is of major importance in medicine. Typical
concentrations of these cations in blood and urine (data in
parantheses) are 143 (125) mM, 5 (65) mM, 1 (4) mM, and
1.5 (4) mM for Na+, K+, Mg++ and Ca++, respectively [2].
Concentration of the lithium cation in plasma is estimated
as 0.09–0.15 μmol/l [4]. On the other hand, barium salts are
known as toxic agents (classic symptoms of barium toxicity
are repeated profound hypokalemia, cardiac arrhythmias,
respiratory failure, prolonged gastrointestinal dysfunction,
paralysis, myoclonus, hypertension, and profound lactic
acidosis) [5], the maximal concentrations of this cation in
serum is in range 3–20 μg/dl [6].
.
.
.
Keywords Fluorescence Sensor Excimer Quantum
chemical calculations
In the last years much attention has been paid to
developing fluorescence sensors.
:
M. Mac (*) T. Uchacz
The working principle of the previously investigated
systems is as follows. The molecules which contain
electron donor and acceptor moieties (so called EDA
systems) are linked by the methine subunit. The donor part
of the molecule is the called recognition moiety. In the
ground state these two parts are almost non-interactive.
Upon excitation to the first singlet state an effective transfer
of one electron from the donative part of the molecule to
the azaromatic skeleton occurs. In consequence the fluo-
rescence of the dye is strongly quenched. However, the
recognition moiety (electron donor part of the molecule)
can act in the ground state as a Lewis base. Thus, upon
Faculty of Chemistry, Jagiellonian University,
Ingardena 3,
30-060 Krakow, Poland
e-mail: mac@chemia.uj.edu.pl
:
:
A. Danel K. Danel E. Kulig
Department of Chemistry, University of Agriculture,
Balicka 122,
31-149 Kraków, Poland
P. Kolek
Institute of Physics, Rzeszow University,
Rejtana 16a,
35-959 Rzeszow, Poland

376
J Fluoresc (2011) 21:375–383
addition of the Lewis acid to the EDA system a complex is
formed and the electro-donative property of this part of
molecule is strongly diminished. Therefore, quenching of
the fluorescence is weaker in the presence of cations and
the system emits the fluorescence light. In consequence, the
fluorescence intensity depends on the cation concentration,
making these compounds usable for qualitative determina-
tion of the analyzed cations.
In our paper we would like to discuss the fluorometric
data on a novel compound being derivative of 1,3,4-
triphenyl-1H-pyrazolo[3,4-b]quinoline.
Experimental
The Synthesis of P1 Molecule
It should be noted that the working principle for some
other molecular systems might be different than that
presented above. There is for instance a group of sensors
containing polycyclic aromatic hydrocarbon fluorophores
(like naphthalene or pyrene) which are linked by crown
ether or open-chain structure (podand). In such compounds
addition of the cations, that are then bounded to the
azacrown moiety, changes the ratio of the excimer-
monomer fluorescence quantum yields [7–9].
It has been established that in many cases the indicators
undergo 1/1 and 2/1 (ligand/metal ion) complexation [10–
12]. Thus, the following equilibria should be taken into
account to describe the overall complexation process.
The synthesis of chelate is depicted in the Scheme 1. 6-
methyl-1,3,4-triphenyl-1H-pyrazolo[3,4-b]quinoline 1 was
prepared by reaction of equimolar amounts p-toluidine,
benzaldehyde and 2,5-diphenyl-2,4-dihydropyrazol-3-one
according to literature procedures [13]. The bromination
of this compound with NBS yielded 6-bromomethyl-1,3,4-
triphenyl-1H-pyrazolo[3,4-b]quinoline 2 [1]. The nucleophilic
substitution of bromomethyl group with di-(2-picolyl)amine
in acetonitrile gave compound 3.
Bis-(pyridin-2-yl-methyl)-(1,3,4-triphenyl-1H-pyrazolo
[3,4-b]quinolin-6-ylmethyl)-amine 3
K1
Mþ þ L Ð MLþ
6-Bromomethyl-1,3,4-triphenyl-1H-pyrazolo[3,4-b]quino-
line 2 (490 mg, 1 mmol), potassium carbonate (140 mg,
1 mmol) and di-(2-picolyl)amine were refluxed together in
acetonitrile (10 ml) for 2 hours. The solution was filtered
and evaporated. The oily residue was dissolved in chloro-
form and filtered through a short pad of alumina. The
K2
þ
Mþ þ 2L Ð ML2
and
K
ML2þ þ Mþ Ð 2MLþ
Scheme 1 a NBS/CCl4 ; b
di-(2-picolyl)amine/K2CO3/
AcCN
Me
b
a
Br
N
N
N
N
N
N
1
2
N
N
N
N
N
N
3

J Fluoresc (2011) 21:375–383
377
product was chromatographed on column packed with
alumina and eluted with toluene:acetone mixture (9:1).
Yellow powder, 300 mg, 50%, mp 205–206 °C.
1H NMR(300 MHz, CDCl3, δppm): 8.55–8.53(m, 2H);
8.44–8.42(m, 2H); 8.15(d, J=8.8 Hz, 1H, 8-H); 7.88(s, 1H, 5-
H); 7.81(dd, J=8.8 Hz, 1.9 Hz, 1H, 7-H); 7.57–7.50(m, 4H);
7.40–7.00(m, 16 H); 3.75(s, 4H); 3.74(s, 2H).
steady-state fluorescence measurements a 365 or 405-nm
line of medium-pressure mercury lamp was used. The
fluorescence quantum yield measurements were carried out
with quinine sulphate in water (Φfl=0.55) [14] as an
actinometer.
The equilibrium constants (K1) for complexation of the
ligand by alkali cations are calculated by fitting the Eq. 1 to
the relative fluorescence intensities in the presence of
different concentration (cM) of the employed alkaline metal
perchlorates [15].
13C NMR(75 MHz, CDCl3, δppm):160.28; 150.87;
149.58; 148.83; 147.36; 144.73; 140.57; 137.00; 135.85;
135.33; 133.26; 132.36; 131.05; 129.78; 129.68; 128.95;
128.53; 128.19; 128.13; 126.61; 126.02; 123.89; 123.32;
122.57; 121.55; 115.60; 60.69; 59.11.
Φð1Þ ꢀ Φð0Þ
ΦðcM Þ ¼ Φð0Þ þ
2cL
Anal. calcd for C41H32N6: C 80.90; H 5.30; N 13.80. C
80.78; H 5.16; N 13.56.
ꢀ
ꢁ
ð1Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ꢁ cL þ cM þ 1=K1 ꢀ ðcL þ cM þ 1=K1Þ ꢀ 4cLcM
MS (ES): (m/z): 609.2 (M+) , 410.2, 332.1.
Melting points were determined on a Mel-Temp Appa-
ratus II (capillary). NMR spectra were recorded on a Varian
(Mercury) spectrometer at 25 °C.
The ab initio calculations were performed using DFT
(B3LYP) method for the ground state and time dependent
perturbation method TD-DFT(B3LYP) for the excited state
with the cc-pVDZ basis set. Computations were performed
with the Gaussian09 program. The optimised geometry of
the ground state was calculated for the investigated ligand
and its complexes with Zn++ and Mg++ cation.
where Φ(0), Φ(∞) and Φ(cM) is the fluorescence intensity of
the dye alone, with the large concentration of the salt and
with the cM concentration of the salt, respectively, cL
indicates the analytical concentration of the ligand.
For 2/1 complexation the binding constant K2 is defined
by the Eq. 2 [1]:
½MLþ2 ꢂ
K2 ¼
ð2Þ
2
½Lꢂ ½Mþꢂ
Fluorescence
The Eq. 1 can be converted into more complex one:
The solvents: cyclohexane (CHX), dibutyl ether (DBE),
butyl chloride (BuCl), ethyl acetate (EtAc), tetrahydrofuran
(THF), dichloromethane (CH2Cl2), methanol (MeOH),
acetonitrile (ACN), dimethylformamide (DMF), and di-
methyl sulfoxide (DMSO) were of spectroscopic grade and
were used as received (all from Aldrich). All the solvents
did not show any traces of fluorescence. For fluorescence
the solutions of the dye were degassed using multiple
freeze-pump-thaw cycles. The sample concentration of the
2x
ΦðcM Þ ¼ Φ0 þ ð Φ1 ꢀ Φ0Þ
ð3Þ
cL
where x denotes the concentration of the complex MLþ2 .
This quantity can be obtained by numerical solving of the
third degree polynomial:
ax3 þ bx2 þ cx þ d ¼ 0
with the following parameters:
ð3aÞ
dyes for spectroscopic measurements was ca. 10−5
M
a ¼ ꢀ4K2
ð3bÞ
(this corresponds to absorbance of ca. 0.2–0.3 at the
excitation wavelengths used for the fluorescence inves-
tigations). Lithium, sodium, barium, magnesium, calcium
(tetrahydrate) and zinc (hexahydrate) perchlorate (Aldrich)
were used as received. In an independent measurement it
was found that a small addition of deionised water
couldn’t influence significantly the fluorescence of the
acetonitrilic solution of the dye. Fluorescence titration
experiments using added salts are performed without
degassing procedure.
b ¼ 4K2ðcL þ cM Þ
2
c ¼ ꢀðK2cL þ 4K2cLcM þ 1Þ
2
d ¼ K2cL cM
The values of the binding constants K1 and K2 were
obtained from fluorescence intensities using nonlinear least-
square fitting coded in FORTRAN.
Results
Fluorescence measurements were made on a home-built
spectrofluorimeter and a time-correlated single photon
counting setup. For time-resolved fluorescence measure-
ments a picosecond diode laser (λ=400 nm, 70 ps pulse
duration) (IBH-UK) was used as the excitation source. For
Absorption spectra of the studied dye are rather insensitive
to solvent polarity. Addition of the studied perchlorate salts
affects the absorption only in the UV spectral range as
pointed out in Fig. 1.

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J Fluoresc (2011) 21:375–383
Table 1 Fluorescence lifetimes (τfl), quantum yields (Φfl) and rate
constants of nonradiative decay (knrad) of P1 in solvents of different
polarities (characterized by dielectric constant εs)
70000
60000
50000
40000
30000
20000
10000
0
Solvent
εs
λmax/nm
Φfl
τfl
knrad/s−1
CHX
2.043
466
467
476
476
478
480
484
482
490
488
488
0.70
0.83
0.70
0.85
0.58
0.63
0.18
0.23
0.11
0.12
0.09
15.70
17.79
17.99
19.22
16.35
18.86
5.46
1.9×107
0.95×107
1.7×107
0.8×107
2.6×107
2.0×107
15.0×107
11.7×107
23.9×107
25.1×107
30.7×107
DBE
3.083
6.08
ETAC
BuCl
7.4
THF
7.52
CH2Cl2
ACE
8.93
21.01
33.00
36.63
38.25
47.24
200 225 250 275 300 325 350 375 400 425 450 475 500
MeOH
ACN
6.61
λ [nm]
3.71
DMF
DMSO
3.50
Fig. 1 Absorption spectra of P1 in acetonitrile without (solid) and
with 0.01 M LiClO4 (dotted) and 0.01 M Zn (ClO4)2 (dashed)
2.97
Fluorescence quantum yields of the dyes depend
strongly on solvent polarity. In solvents of low polarities
the fluorescence quantum yields are very high and decrease
significantly with increasing solvent polarity, even though
the position of the maximum is almost not influenced by
polarity of the solvent.
The fluorescence decay functions are clearly monoexpo-
nential in all employed solvents. In solvents possessing
dielectric constants less that 21 (i.e. less polar than acetone)
the fluorescence lifetimes are rather long and close to the
value characteristic of the parent molecule 1,3,4-triphe-
nyl1H-pyrazolo[3,4-b]quinoline (ca 17 ns). This indicates
that the intramolecular electron transfer, responsible for the
fluorescence quenching, is not operative in solvents of low
and medium polarities. The situation changes in highly
polar solvents such as acetonitrile. In these solvents an
effective quenching is observed. This causes a shortening of
the fluorescence lifetimes and quantum yields as can be
recognized from the data collected in Table 1.
20
0,8
0,6
16
0,4
0,2
12
0,0
0
10
20
30
40
50
εs
8
4
The dependence of these parameters on the solvent
dielectric constants are presented in Fig. 2.
Upon addition of perchlorates to the acetonitrile sol-
utions of the dye P1 the fluorescence significantly
increases. The position of the fluorescence maximum is
not changed due to added salts except in the presence of
Zn++ and Mg++. The dependence of the fluorescence
quantum yield on the salt concentration is presented in
Fig. 3.
0
10
20
30
40
50
εs
3,5x108
3,0x108
2,5x108
2,0x108
1,5x108
1,0x108
The binding constants calculated using eqs. 1 and 2-3 are
collected in Table 2.
5,0x107
Discussion
0,0
The studied molecule does not show a significant solvent
polarity influence as regards both the ground state
absorption band and the position of the fluorescence maxi-
mum. However, the recognition moiety (1-(pyridin-2-yl)-N-
(pyridin-2-ylmethyl)methanamine) influences significantly
the fate of the fluorescing state of the fluorophore.
Fluorescence lifetimes and quantum yields depend on
0
10
20
30
40
50
εs
Fig. 2 Dependence of fluorescence lifetimes of P1 (top), fluorescence
quantum yields (top inset) and nonradiative rate constant (bottom) on
the solvent dielectric constants. The correlation lines are drawn only
for presentation purposes

J Fluoresc (2011) 21:375–383
379
even a small concentration of the cations is sufficient to
complexate the dye by the cation and this complex is
stable in the excited singlet state. It implies a large
sensitivity and good selectivity of this dye to the presence
of the investigated cations as can be recognized from the
different binding constants (Tab. 2). Thus, this dye may be
used as a fluorescing indicator for the cations in
acetonitrile.
An interesting behaviour has been observed upon
addition of the bivalent cations like Mg++ and Zn++.
Contrary to the systems investigated previously (K1 and
L1) [1] addition of these cations causes a significant
change of the spectral distribution in the fluorescence of
the formed complexes as presented in Fig. 4.
52,5
Ca++
Zn++
45,0
Ba++
Li+
Mg++
37,5
30,0
22,5
15,0
7,5
Na+
1E-6
1E-5
1E-4
1E-3
log(c)
0,01
0,1
These complexes have a stoichometry 2/1 as can be
judged from the salt concentration dependence of the
integral intensity of the fluorescence. The question which
remains to be addressed is to explain the reason of the
spectral shift of the fluorescence band in the presence of
Zn and Mg cations. We postulate that the factor
responsible for the small fluorescence shift in the
presence of these cations is a formation of an intramo-
lecular excimer- like product.
The red shift in the fluorescence spectrum of P1 in the
presence of Zn++ in acetonitrile is about 17 nm, while the
half-with of the fluorescence band is enlarged by ca. 15 nm
(94 vs 109 nm). Similar changes (although less pro-
nounced) occur in the presence of Mg++. The fluorescence
lifetime of the complex P1 with Zn++ measured at different
observation wavelengths (i.e. at the blue and red side of the
fluorescence spectrum) differ one from another but the
difference is rather small (10.7 vs 12.1 ns).
Fig. 3 Salt concentration dependence (in logarithmic scale) of the
relative fluorescence quantum yield of P1 upon addition of lithium (black
circles), sodium (open diamonds), calcium (black squares), barium
(black triangles), magnesium (open circles) and zinc perchlorates (stars)
in acetonitrile. The correlation lines are calculated either from eq. 1 (Li+,
Na+, Ba++ and Ca++) or eqs 2–3 (Mg++ and Zn++
)
solvent polarity as can be predicted from the electron transfer
theory. In the excited state one electron from the donative
part of the molecule is transferred to the acceptor part,
forming the nonfluorescing monomolecular radical pair
which recombines recovering the ground state of the system.
Increasing of the solvent polarity decreases the energy of the
charge transfer pair, which is formed in monomolecular
charge transfer process, resulting in more efficient fluores-
cence quenching in polar solvents. In solvents of low and
medium polarity the quenching is hardly observed but
already in acetone the significant reduction of these
parameters is observed. These observations may be useful
in designing this molecule as a fluorescence indicator of
small inorganic cations.
Excimer Formation
The change of the first absorption band upon addition
of the salts is not significant. Only small influence in the
deep UV-region is observed (cf. Fig. 1). Addition of
small amounts (up to 1×10−1 M) of the perchlorates
(lithium, sodium, calcium, barium, magnesium and zin-
cum) to the solution of P1 in acetonitrile causes a ca. 4–7-
fold increase of the fluorescence intensity. It means that
If the excited molecule (M*) undergoes a collision with the
same but not excited molecule (M) an excited dimer is formed
(MM)*. The formation of the bimolecular excimers is a
diffusional process, therefore the formation is strongly prefer-
able at higher molecule concentrations because this process
proceeds within the fluorescence lifetime of the molecule.
Table 2 Binding constants
Cation(X)
Radius (Å) [16]
Charge density of cation [17]
K1(P1M+n)/M−1 or K2(P12M+n)/M−2
i.e. equilibrium constants
for complexes between the
compound P1 and different
inorganic cations in acetonitrile
determined from fluorimetric
titration with the estimation
errors in parentheses
Li+
Na+
Ba++
Ca++
Mg++
Zn++
0.59
1.02
1.34
1.0
1.47
1.03
1.49
2.02
3.03
2.7
K1=3000(3%)
K1=45.3(2%)
K1=5200(2%)
K1=1.5×105(5%)
K2=2.6×109 (3%)
K2=2.5×1011(5%)
0.66
0.74

380
J Fluoresc (2011) 21:375–383
1,0
0,8
0,6
0,4
0,2
0,0
structure. When the ground state complex (MM) already
exists then displacement of the two molecules to achieve a
sandwich configuration appropriate for the excimer fluo-
rescence occurs very quickly. In such cases the rise time of
the excimer fluorescence could not be detected. Such type
excimers, where an excimer emission appears or disappears
in the presence of ions, are commonly used as fluorescing
ion indicators [20].
To check the possibility of existence of the excimer-like
form in our systems we decided to perform some theoretical
investigations on our systems and the excimer consisting of
two simpler 1H-pyrazolo[3,4-b]quinoline molecules (PQ).
This molecule resembles P1 but contains smaller number of
atoms which renders the quantum chemical calculations in
the excited state possible.
(P1)2Mg++
(P1)2Zn++
P1- bare
400
450
500
550
600
650
700
λ/nm
Fig. 4 Fluorescence spectra of P1 (solid line) and that of 2/1
complexes of P1 with Zn (dotted line) and Mg (dashed line) in
acetonitrile
Quantum Chemical Calculations
The computed vertical excitation energy for the ligand
molecule (P1) is 2.87 eV and the relaxed fluorescence energy
is 2.44 eV (Table 3). These values are very close to excitation
energies for the bare fluorophore (P2) without the recognition
unit (1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine),
proving that this group does not take part in the excitation.
Large size of the investigated complexes causes that the
vertical excitation energies and the energy of fluorescence
from the relaxed excited state could be calculated only for the
single fluorophore (P1). The fluorescence spectral shift
caused by the excimer formation between ligands of 2:1
complexes were estimated by performing computations for a
simpler model molecule. For this purpose, 1H-pyrazolo[3,4-
b]quinoline molecule (PQ) was chosen as the fluorophore and
It is well-known that many aromatic and planar
molecules (such as naphthalene and anthracene) form
excimers. This manifests itself by existence of the addi-
tional, broad and structureless fluorescence band of MM*
located at wavelengths higher than that of monomer M*. In
some cases these bands are well resolved. The intensity of
the excimer fluorescence grows up when the concentration
of M increases. In such excimers the time evolution of the
fluorescence of the monomer and the excimer are different.
The decay of the monomer fluorescence (M*) is the sum of
two exponents whereas in the case of the excimer
fluorescence (MM*) the preexponential factors have oppo-
site signs. It means that the rise and the decay of the
excimer fluorescence is observed.
When two monomers are linked by a flexible chain the
intramolecular excimer may be also formed [18, 19],
Formation of the excimer involves not a translational
motion but a rotational one to form the sandwich-type
N
N
H
N
Table 3 Excitation energies of the ligand and model molecules as
computed with TD-DFT(B3LYP)/cc-pVDZ
Molecule
ΔEvert /eV
ΔErelaxed fluo /eV
Me
P1
2.87
2.89
3.55
3.54
–
2.44
2.46
3.17
2.47
3.04
3.09
3.14
3.16
3.17
P2
N
PQ
N
N
PQ·PQ(Req) or PQ·PQ*(Req*)
PQ·PQ*(R=4.77Å)
PQ·PQ*(R=4.98Å)
PQ·PQ*(R=5.33Å)
PQ·PQ*(R=5.67Å)
PQ·PQ*(R=6.01Å)
–
–
Scheme 2 Chemical structures of the molecules characterized in
Table 3. Top—PQ (1H-pyrazolo[3,4-b]quinoline), bottom P2
(6-methyl 1,3,4-triphenyl-1H-pyrazolo[3,4-b]quinoline)
–
–

J Fluoresc (2011) 21:375–383
381
should not modify the spectroscopic properties of the
complex.
The ground state geometry of the dimer PQ·PQ was
optimised for the distance between monomers of ca. 10.5Å
to 11.8Å (the NN’ distances) where the distance between
the relevant nitrogen atoms of both molecules is larger by
0.7Å than the distance between the carbon atoms at the
opposite end of the molecules. Thus, this system may be
treated as consisting of isolated molecules. The optimised
geometry of the excited state, however, points out to the
excimer formation with much smaller equilibrium distance
(Req) between monomers equal ca. 3.4Å (CC′=3.385Å,
NN′=3.498Å, see Fig. 5).
The relaxed fluorescence energy for the excimer
PQ·PQ* is 2.47 eV while for the isolated monomer the
energy 3.17 eV is predicted. This strong red-shift is as
large as −0.7 eV and it overwhelms the experimental
fluorescence red-shift which is only −0.09 eV for the
P12Zn++ complex and −0.06 eV for the complex with Mg++
cation. The above results show that the fluorescence red-shift
could be extraordinarily large if the PQ molecules would
interact optimally and both molecular planes were close to
each other. This is certainly not the case of our systems
P12Zn++ and P12Mg++.
Fig. 5 PQ·PQ* excimer. Lines mark distances: CC′=3.385Å and
NN′=3.498Å
its excimer consisting of two PQ molecules was examined
(see Scheme 2).
The influence of the phenyl groups on the spectroscopic
properties of the system was investigated by comparison
with 6-methyl 1,3,4-triphenyl-1H-pyrazolo[3,4-b]quinoline
(P2). Although the presence of amino group (in P1) causes
the existence of the CT state, the charge transfer cannot
occur if the nitrogen atom lone pair is used for complex-
ation with a metal cation. Thus, the neglected substituents
Fig. 6 Optimised structures
of the P12Zn++ (bottom)
and P12Mg++ (top) in the
ground state

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J Fluoresc (2011) 21:375–383
Fig. 7 Hypothetical (unopti-
mised) structure of excimer
P12Zn++* obtained by rotation
around the N-CH2 bonds linking
the fluorophores with the core
complex
The optimised structures of the complexes in the ground
state are shown in Fig. 6. The most important effect of the
substituent groups (phenyl rings) which should be here
taken into account is the sterical hindrance that they cause.
The phenyl substituents at positions 3 and 4 in the ground
state are twisted with respect to the fluorophore plane by 46
and 68 degrees in, respectively. These angles are expected
to decrease in the excited state but only slightly. Thus, the
optimal distance between the phenyl rings of the two
monomers of about 3.4Å causes that the fluorophores could
not approach closer each other than 4.8Å.
parallel and the rotation with respect to the CH2-fluorophore
bonds to align the fluorophores in nearly parallel planes. The
possible conformation of the P12Zn++ excimer is presented
in Fig. 7. Thus, the minimal distance between the 1,3,4-
triphenyl1H-pyrazolo[3,4-b]quinoline fluorophores in the
real excimer is limited by the minimal distance between the
two carbon atoms of the -CH2- bridges linking the
recognition moiety (1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)
methanamine) with the 1,3,4-triphenyl1H-pyrazolo[3,4-b]
quinoline. The Mg-Namino and Zn-Namino bond length in
the ground state are equal 2.30Å. Taking into account the
linking N-CH2 bonds, the parts of the fluorophores
connected to this carbon atoms should not approach each
other closer than roughly 5.5Å if the core complex and the
-CH2- bridges are assumed to be rigid. This rather large
The excimer formation in the complexes P12Zn++ and
P12Mg++ is facilitated by conformational change concerning
primarily the rotation of the fluorophore groups around the
Namino-CH2 bonds to make the long axis of the fluorophores
Fig. 8 Distance dependence
of the fluorescence energies of
the PQ·PQ* excimer. Open
circles mark the energy of the
excimer calculated for frozen
geometry of monomers taken
from the equilibrium structure
(distance 3.385Å), full circles
mark energy of the optimised
excimer. For more detailed
explanation see text above

J Fluoresc (2011) 21:375–383
383
separation between the fluorophore’s planes seems to be
responsible for only small bathochromic shift of the
excimer fluorescence as compared to that for the bare P1
molecule.
The enhanced sensitivity and selectivity for Zn++ and
Mg++ arises from an efficient complexation of these cations
with two P1 molecules.
Moreover, the remarkable bathochromic shift in fluores-
cence (17 nm for P12Zn++ and 13 nm for P12Mg++
complex) is observed. This spectral feature is most
probably due to excimer formation. This is partially
supported by the quantum chemical calculations.
To illustrate the dependence of the spectral shift of
fluorescence on the distance between the fluorophores the
excitation energy curve was calculated (see Fig. 8) for
PQ·PQ*. The open circles mark the fluorescence energy
calculated for the excimer geometries obtained from the
optimised geometry by increasing the distance between the
monomers without reoptimisation. Therefore this curve is
relevant for distances close to the minimum energy point but
at large distances the calculation predicts too high energies
and false limit for isolated fluorophores. To overcome this
deficiency and to give a more reliable estimation of the
fluorescence red-shift at the distances available for the
P12Zn++ and P12Mg++ excimers the geometries of PQ·PQ*
were reoptimised for frozen few values of the CC′ distance
marked in Fig. 5. The CC′ distances and the fluorescence
energies are collected in Tab.3 and graphically presented in
Fig. 8 (full circles). A good agreement with the experimental
red-shift for the P12Zn++ complex (ca. 0.08 eV) is achieved
at the distance roughly 5Å. At this distance the value of the
fluorescence energy for the excimer PQ.PQ* is equal
3.09 eV while for the isolated molecule PQ* it is 3.17 eV .
For smaller distances the fluorescence energy decreases
rapidly. On the other hand, for distances larger than 5.5Å the
fluorescence energy is essentially the same as for the isolated
fluorophore. Finally, it should be noticed that the computa-
tions with larger basis sets and with correction for Van der
Waals dispersion interaction could predict an effective
excimer formation and a significant red-shift fluorescence
at slightly larger distances than the TD-DFT(B3LYP)/cc-
pVDZ predictions discussed above.
Acknowledgments The authors would like to thank Dr Andrzej M.
Turek for editorial comments. Quantum chemical calculations were carried
out in the Academic Computer Center “Cyfronet” in Krakow, Poland
(supported by Grant No. MNiSW/IBM_BC_HS21/UJ/075/2009).
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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Conclusions
A novel fluorescent sensor for determination of small
inorganic cations was synthesized and investigated by
means of fluorescence spectroscopy and quantum-
chemical calculations. The stability of the fluorescent
complexes of P1 with inorganic cations (Mn+) (P1Mn+
and P12M n+) were determined by fluorescence titration.
This indicator shows a qualitatively good selectivity for
small inorganic cations and good sensitivity especially
towards bivalent cations such as Zn++ and Mg++. This
system may be applicative in practice for determination of
these cations in the presence of other ones.