Synthesis and photophysical properties of a fluorescent TREN-type ligand incorporating the coumarin chromophore and its zinc complex

Article abstract of DOI:10.1016/j.tetlet.2005.04.059

A new, UV-excited fluorescent Zn²⁺ indicator was synthesized and the spectral profile of its free and Zn²⁺ bound forms were studied. The fluorescent properties of this probe are due to the 7-amino-4-methylcoumarin fluorophore, which is conjugated with the tris(2-aminoethyl)amine (TREN) that functions as the zinc-chelating moiety. The compound exhibits a Zn²⁺ dissociation constant of 18.0 μM. The fluorescence spectra of the probe showed a clear shift in excitation wavelength maxima upon Zn²⁺ binding, indicating its potential use as ratiometric Zn²⁺ indicator.

Full text of DOI:10.1016/j.tetlet.2005.04.059

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4193–4196
Synthesis and photophysical properties of a fluorescent TREN-type
ligand incorporating the coumarin chromophore and
its zinc complex
Marianna Dakanali,a Emmanuel Roussakis,a Alan R. Kayb and
Haralambos E. Katerinopoulosa,*
aDivision of Organic Chemistry, Department of Chemistry, University of Crete, Heraklion 71 409, Crete, Greece
bDepartment of Biological Sciences, 138 BB, University of Iowa, Iowa City, IA 52242, USA
Received 8 March 2005; revised 13 April 2005; accepted 13 April 2005
Available online 29 April 2005
Abstract—A new, UV-excited fluorescent Zn2+ indicator was synthesized and the spectral profile of its free and Zn2+ bound forms
were studied. The fluorescent properties of this probe are due to the 7-amino-4-methylcoumarin fluorophore, which is conjugated
with the tris(2-aminoethyl)amine (TREN) that functions as the zinc-chelating moiety. The compound exhibits a Zn2+ dissociation
constant of 18.0 lM. The fluorescence spectra of the probe showed a clear shift in excitation wavelength maxima upon Zn2+ binding,
indicating its potential use as ratiometric Zn2+ indicator.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Zn2+ is the second most abundant transition metal, after
iron, in biological systems.1 Since the 1940s, there
has been a steady stream of data implicating Zn2+ in a
number of biological processes. Zinc may serve as a
structural element in enzymes and transcription factors,
as well as at the catalytic site of a number of enzymes.
The interest in Zn2+ is compounded by its involvement
in a number of neuropathologies such as AlzheimerÕs
disease (AD), amyotrophic lateral sclerosis (ALS), Par-
kinsonÕs disease, hypoxia–ischemia, and epilepsy.2
soning behind the design of such molecules have been
published and, as a result, a host of fluorescent zinc
probe syntheses have been reported and reviewed in
recent articles.4–8
In this letter, we describe the synthesis and the fluores-
cence spectra of a new chemosensor for zinc 1, where
the 7-amino-4-methylcoumarin fluorophore and the
tris(2-aminoethyl)amine (TREN) as zinc-chelating moi-
ety have been conjugated. The choice of the fluorescent
molecule 1 as a zinc probe was based on the following
rationale: depending on the nature of the photo-induced
process, fluorescent ion probes with a linked fluoro-
phore and ionophore can be classified into two types:
photo-induced electron transfer (PET) and photo-in-
duced charge transfer (PCT) probes.9 In PET sensors,
upon excitation of the ion free form, a donor–acceptor
electron transfer causes fluorescence quenching. When
the sensor is bound to a target cation, such transfer is
prevented and the net result is a fluorescence enhance-
ment. In PCT sensors, the fluorophore contains an elec-
tron-donating group (in this case the 7-amino group)
conjugated to an electron-withdrawing group (in this
case the coumarin carbonyl), which upon excitation,
undergoes internal charge transfer (ICT) from the donor
to the acceptor.10 Coordination of the target ion with
the electron-donor moiety destabilizes the system result-
ing in a hypsochromic shift in its excitation spectrum.
Zn2+ is often referred to as the Ôsilent ionÕ since, unlike
other biological transition metal ions (such as Fe2+
,
Mn2+, or Cu2+), it does not have an intrinsic spectro-
scopic or magnetic signal because of its 3d10 4s0
electronic configuration. However, its ability to form
tetracoordinated complexes with a variety of organic
moieties that can be connected to a fluorophore can be
used as the basis for synthesizing fluorescent probes that
may be useful in biological systems.3
The design and synthesis of fluorescent zinc probes re-
quires the combined expertise of chemistry, biology,
and medicine. A number of articles analyzing the rea-
*
Corresponding author. Tel.: +30 2810 393 626; fax: +30 2810 393
601; e-mail: kater@chemistry.uoc.gr
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.04.059

4194
M. Dakanali et al. / Tetrahedron Letters 46 (2005) 4193–4196
Such a shift is highly desired in fluorescence ion probes,
since it offers the possibility of ratiometric measure-
ments of ion concentrations. Given that in 1, one of
the nitrogens is part of both fluorophore and chelator,
the probe is expected to belong to the latter type and
its excitation spectrum should undergo a blue shift upon
zinc ion binding.
1, the Zn2+ free probe exhibits an excitation maximum
wavelength at 359 nm, which shifts to 337 nm upon zinc
binding. This set of spectra has an isosbestic point at
340 nm. When excited at 359 nm the molecule shows
The stepwise synthesis of 1 is depicted in Scheme 1. 7-
Amino-4-methylcoumarin, protected as tosyl amide 2,
was reacted with tris(2-chloroethyl)amine hydrochloride
in the presence of Cs2CO3 to yield dichloride 3. Depro-
tection of the amine via treatment with H2SO4 and con-
version of the dichloride 4 to the corresponding diazide
5, followed by catalytic reduction using 10% Pd/C as
catalyst, furnished the desired TREN analog 1 in 18%
overall yield.11
A stock indicator solution was prepared in nanopure
water at 0.02 M concentration. An aliquot of this stock
solution was then added to a 10 mM HEPES buffer,
containing 140 mM NaCl and 2.5 mM KCl, to make a
final indicator concentration of 10 lM, and the pH
was adjusted to 7.8. Zn2+-containing solutions were
prepared by adding Zn2+ (0.02 M ZnSO4 solution pre-
pared in nanopure water) to the final indicator solution,
to give Zn2+ concentrations ranging from 1 to 500 lM.
Figure 1. Excitation (A) and emission (B) spectra of 10 lM TREN-
type probe 1 as a function of increasing Zn2+ concentration in 10 mM
HEPES buffer, containing 140 mM NaCl and 2.5 mM KCl, adjusted to
pH 7.8. Spectra were obtained at 22 ꢁC in an Aminco Bowman Series 2
fluorimeter with excitation and emission slit widths set at 4 nm. The
emission and excitation wavelengths were set at 450 and 359 nm,
respectively.
The spectral profile of probe 1 was studied in solutions
of increasing Zn2+ concentration. As shown in Figure
CH3
CH3
TsCl
pyridine, CH2Cl2
rt, 18 h, 78%
O
O
NHTs
O
O
NH2
2
Cl
Cl
HCl
N
Cs2CO3,, CH3CN
80 oC, 18 h, 80%
Cl
CH3
CH3
O
Cl
Cl
Cl
N
N
O
O
N
O
N
H
c. H2SO4
Ts
90 oC, 1 h, 86%
3
4
Cl
LiN3, DMF
60 oC, 6 h, 35%
CH3
O
CH3
NH2
NH2
N3
N
N
O
N
H
H2, 10% Pd/C
O
O
N
H
MeOH, rt, 2 h, 95%
1
N3
5
Scheme 1.

M. Dakanali et al. / Tetrahedron Letters 46 (2005) 4193–4196
4195
an emission maximum at 450 nm, which does not shift
even at saturation Zn2+ levels. However, a 42% decrease
in fluorescence intensity was observed upon addition of
300 lM Zn2+. When excited at 337 nm, the fluorescence
spectra of the probe solutions exhibit an emission maxi-
mum at 450 nm and a small increase in fluorescence
intensity as the concentration of Zn2+ increases. From
the hypsochromic shift and the change in excitation
intensity observed, it appears that compound 1 is
indeed a PCT-type probe suitable for ratiometric mea-
surements.
The dissociation constant value, calculated according to
TsienÕs algorithm for ratiometric dyes,12 was in the
micromolar region (Kd = 18 lM) and comparable to
those reported for cyclen-type sensors ZnACF-1,
ZnACF-2,13 RF-2,14 a di(2-picolyl)amine analog of
6,7-dimethoxy-4-methylcoumarin,15 and a cyclen-coup-
led analog of 6,7-dimethoxy-4-methylcoumarin.16 How-
ever, the probe has considerable lower affinity than
anthracene- or dansyl-coupled polyazamacrocyclics.4
Apparently, conjugation of the free electron pair of
the 7-amino group with the coumarin system reduces
its availability for coordination with Zn2+, thus reducing
its affinity to the ion as compared to a calculated Kd
value of 1.9 · 10ꢀ10 M for TREN.17 A tridansylated
TREN as well as a tris(4-dimethylaminobenzyl)-TREN
probe have been reported in the literature, but Kd values
were not calculated in these studies.18,19
Figure 2. Excitation (A) and emission (B) spectra of 10 lM TREN-
type probe 1 as a function of pH in 10 mM HEPES buffer, containing
140 mM NaCl, 2.5 mM KCl, and 300 lM Zn2+. The pH was adjusted
by addition of HCl solution. Spectra were obtained at 22 ꢁC in an
Aminco Bowman Series 2 fluorimeter with excitation and emission slit
widths set at 4 nm. The emission and excitation wavelengths were set at
450 nm and 359, respectively.
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Given that aliphatic polyamine sensors are susceptible
to protonation, we examined the spectral profile of the
free and zinc-bound forms at pH 6.0–8.0. An 8% reduc-
tion of fluorescence was observed when the pH was
increased from 6.0 to 7.0 in solutions of the zinc-free
sensor. The corresponding decrease in fluorescence,
when the pH value was further increased from 7.0 to
8.0, was 4%. No shift in either excitation or emission
maxima was observed (data not shown).
In contrast to the profile exhibited by the free probe, the
ion-bound form undergoes drastic changes upon the
same increase in pH values. A total fluorescence de-
crease of 41% was observed and, at a pH range 6.0–
7.0, the excitation spectrum is identical to that of the
free probe (Fig. 2). However, increasing the pH from
7.0 to 8.0 results in a change of the excitation spectrum
to that of the bound form. As suggested by the excita-
tion spectra profile, zinc binding is pH dependent since
the sensor is protonated in acidic to neutral media and
Zn2+ is able to coordinate with the TREN system at
pH values above 7.5. Similar arguments have been used
to explain the fluorescence profile of the other two
TREN-type zinc probes in solutions of decreasing
acidity.15,20
0.0
Metal Ion
Figure 3. Mn+-selectivity profile of TREN-type sensor: Bars represent
ratio integrated fluorescence response ꢂF337 nm/ꢂF359 nm, where 337 and
359 nm are the kexc max of the zinc-bound and the free dye, respectively.
Emission was integrated between 370 and 650 nm. Spectra were
acquired in 10 mM HEPES buffer, containing 140 mM NaCl and
2.5 mM KCl, adjusted to pH 7.8 at 25 ꢁC. Gray bars: Aliquots of
concentrated stock solutions of each metal ion were added to the
10 lM dye solution to provide 10 lM total metal ion concentration,
and the fluorescence response was calculated. Black bars: Zn2+
(10 lM) was added subsequently to the solution containing the metal
ion, and the response was measured.
ratio in the presence of zinc is shown (black bar), as
compared to that of the free dye (gray bar). Such an
increase in the fluorescence ratio is expected, since addi-
tion of zinc increases F337 while decreasing F359. The rest
of the measurement sets indicate the ratio of fluores-
cence of the dye in the presence of 1 equiv of metal (gray
bars) versus the ratio of fluorescence after addition of
1 equiv of zinc to the sample (black bars). The results
The potential of this compound for use as a ratiometric
dye in biological systems is shown in an ion competition
study of a range of metals binding to the sensor. Sam-
ples were excited at kexc free (359 nm) and kexc bound
(337 nm) and the fluorescence ratio was calculated, inte-
grating the emission between 370 and 650 nm (Fig. 3). In
the first set of measurements the increase of fluorescence

4196
M. Dakanali et al. / Tetrahedron Letters 46 (2005) 4193–4196
6. Kikuchi, K.; Komatsu, K.; Nagano, T. Curr. Opin. Chem.
Biol. 2004, 8, 182–191.
7. Lim, N. C.; Schuster, J. V.; Porto, M. C.; Tanudra, M. A.;
of this study are very similar to those published for zinc
fluorescent sensor RF2.14 Binding by these metal ions
appears to be weak, since the presence of metals does
not reduce the fluorescence ratio of the dye and subse-
quent addition of Zn2+ to these TREN-probe solutions
enhances fluorescence. As expected, the latter effect is
cancelled in the case of paramagnetic Ni2+ and Cu2+
ions due to their fluorescence quenching properties.
Yao, L.; Freake, H. C.; Bruckner, C. Inorg. Chem. 2005,
¨
44, 2018–2030.
8. Lim, N. C.; Freake, H. C.; Bruckner, C. Chem. Eur. J.
¨
2005, 11, 38–49.
9. Valeur, B.; Leray, I. Coord. Chem. Rev. 2000, 205, 3–
40.
10. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice,
T. E. Chem. Rev. 1997, 97, 1515–1566.
In conclusion, we have synthesized a new UV-excited
fluorescent TREN-type Zn2+ indicator incorporating
the coumarin chromophore, and studied the spectral
profile of its free and Zn2+ bound forms. The compound
exhibits a Zn2+ dissociation constant of 18.0 lM. The
fluorescence spectra of the probe showed a clear shift
in excitation wavelength maxima upon Zn2+ binding,
indicating its potential use as a ratiometric zinc
indicator.
11. Selected data. Compound 1: 1H NMR (CDCl3, 500 MHz):
d 7.27 (d, J = 9 Hz, 1H, Ar), 6.53 (br d, J = 9.0 Hz, 1H,
Ar), 6.43 (br s, 1H, Ar), 5.91 (br s, 1H, CH3–C@CH), 3.18
(br t, J = 5.5 Hz, 2H, NHCH2), 2.79 (t, J = 5.5 Hz, 4H,
CH2NH2), 2.73 (t, J = 5.5 Hz, 4H, NCH2), 2.58 (t,
J = 5.5 Hz, 2H, CH2N), 2.29 (s, 3H, CH3). ESI-MS
(positive ion mode): m/z 305 [M+H]+; ESI-ion trap MSn
(positive ion mode): MS2 m/z 305!288 [M+HꢀNH3]+,
MS3 m/z 288!271 [Product ion(288)ꢀNH3]+, MS4 m/z
271!214 [Product ion(271)ꢀ2C2H4ꢀHÅ]+, MS5 m/z
214!186 [Product ion(214)ꢀCO]+, MS6 m/z 186!158
[Product ion(186)ꢀC2H4]+.
Acknowledgements
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6816–6823.
This project was supported by grants from the EPEAEK
program of the Greek Ministry of Education and
funded in part by a grant from NINDS (to A.R.K.).
The authors thank Professor S. Pergantis at the Univer-
sity of Crete, for his help with the ESI/MS experiments.
15. Lim, N. C.; Bruckner, C. Chem. Commun. 2004, 1094–
¨
1095.
16. Lim, N. C.; Yao, L.; Freake, H. C.; Bruckner, C. Bioorg.
Med. Chem. Lett. 2003, 13, 2251–2254.
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