A novel fluorescein-based dye containing a catechol chelating unit to sense iron(III)

Article abstract of DOI:10.1016/j.dyepig.2011.10.010

In the present work we report the synthesis and characterization of a novel fluorescein-based dye containing a catechol chelating unit designed to act as a chemosensor for iron(III). The compound was prepared using conventional and microwave-assisted amid

Full text of DOI:10.1016/j.dyepig.2011.10.010

Dyes and Pigments 93 (2012) 1447e1455
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A novel fluorescein-based dye containing a catechol chelating unit to sense
iron(III)
,
b
b
b
a,
*
Carla Queirós a, Ana M.G. Silva b , Sílvia C. Lopes , Galya Ivanova , Paula Gameiro , Maria Rangel
*
a REQUIMTE, Instituto de Ciências Biomédicas de Abel Salazar, 4099-003 Porto, Portugal
b REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work we report the synthesis and characterization of a novel fluorescein-based dye
containing a catechol chelating unit designed to act as a chemosensor for iron(III). The compound was
prepared using conventional and microwave-assisted amide coupling of a catechol derivative with
a 5(6)-carboxyfluorescein scaffold. One of the most interesting features of this conjugate structure is
a high affinity binding site on the catechol ring close to the fluorophore. As a consequence, the behaviour
of the new compound in the presence of metal ions of biological importance was monitored by optical
spectroscopy (at physiological pH). The results show that the conjugate is particularly sensitive to
iron(III), thus suggesting that it may be used as fluorescent chemosensor for the detection of iron(III) in
cellular systems.
Received 27 June 2011
Received in revised form
25 September 2011
Accepted 9 October 2011
Available online 20 October 2011
Keywords:
Catechol
Fluorescein
Chemosensor
Fluorescence spectroscopy
Iron(III)
Ó 2011 Elsevier Ltd. All rights reserved.
Microwave irradiation
1. Introduction
scaffold in order to supply a selective and sensitive receptor site for
each metal ion [13]. Specifically, we are focusing our attention on
There is an increasing interest in the design of fluorescent
chemosensors to detect biologically important metal ions, such as
zinc(II), iron(III) and copper(II) [1e4]. According to Czarnik, a fluo-
rescent chemosensor is a conjugate incorporating a binding site,
a fluorophore (fluorescent dye) and a mechanism that allows
communication between the two [5]. Based on this concept, fluo-
rescent chemosensors have been widely used in the construction of
fluorescence devices for chemical, biological and medicinal appli-
cations [6,7]. These fluorescence devices are an alternative method
to laborious analytical techniques, since they allow not only the in
situ and real time detection but also analyte quantification at lower
cost [8].
Xanthenes, for example fluorescein, are one of the most common
classes of fluorophores used for iron(III) [9], copper(II) [10] and
zinc(II) [11] fluorescent chemosensors. This fact is due to the
excellent photophysical properties of fluorescein namely, high
molar absorptivity, intense fluorescent spectrum in the visible
region, high quantum yield and photostability [12]. In addition,
different functionalities can be introduced on the fluorescein
catechol derivatives as receptors mainly because of their intrinsic
acidebase properties (pKa ¼ 9.2 and 13.0 for catechol in water) [14].
Catechol-containing molecules are of widespread biological occur-
rence and importance [15]. It is worth to mention that catechol
groups are the chelating moieties of various siderophores and make
up the main structure of catecholamines. The latter compounds are
of pharmacological use in fields such as: Parkinson’s disease treat-
ment [16] and hypertension [17] in addition to their physiological
action as neurotransmitters. Catecholate compounds can also be
used in breast cancer therapies [18].
Iron represents an essential trace element holding a central
position in virtually all organisms as the physiologically most
abundant and versatile transition metal [19]. Taking into account
the biological relevance of iron and the need to access the element
within the cell a series of fluorescent iron(III) chelators bearing
a
fluorescein function covalently bound to a 3-hydroxy-4-
pyridinone unit have been synthesised [20,21]. Some of these
fluorescent iron chelators were used to determine labile iron in
primary hepatocytes [22,23].
Here, we describe the synthesis and photophysical properties of
a fluorescein-based dye containing a catechol chelating unit (Cat1,
Scheme 1) close to the fluorophore. The catechol unit exhibits a very
high affinity for iron(III) and consequently this newly prepared
conjugate can be used as a highly efficient chemosensor for iron(III).
* Corresponding authors.
E-mail addresses: ana.silva@fc.up.pt (A.M.G. Silva), mcrangel@fc.up.pt
(M. Rangel).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.10.010

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C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
Scheme 1. Synthetic approach to synthesise conjugate 5.
2. Experimental
under argon atmosphere. After stirring at 0 ꢀC for 15 min, the
reaction mixture was allowed to warm up to room temperature and
the stirring was maintained for 4 h. After that time, the reaction
mixture was precipitated into an iceewater mixture and neutral-
ized using citric acid. The white solid obtained was filtered, washed
with water, dissolved in chloroform and crystallized in n-hexane to
2.1. Materials and instruments
Reagents and solvents were purchased as reagent-grade and
used without further purification unless otherwise stated.
NMR spectra were recorded on a Bruker Avance III 400, oper-
ating at 400.15 MHz for protons and 100.62 MHz for carbons,
equipped with pulse gradient units, capable of producing magnetic
field pulsed gradients in the z-direction of 50.0 G/cm. NMR spectra
were acquired in deuterated methanol or deuterated chloroform at
300 K. Two-dimensional 1H/1H correlation spectra (COSY), gradient
selected 1H/13C heteronuclear single quantum coherence (HSQC)
and 1H/13C heteronuclear multiple bond coherence (HMBC) spectra
were acquired using the standard Bruker software. Mass spectra
were acquired by Unidade De Espectrometria De Masas of Santiago
de Compostela and microanalyses were acquired by Unidad De
Análisis Elemental of Santiago de Compostela. Flash chromatog-
raphy was carried out using silica gel Merck (230e400 mesh).
Electronic absorption spectra were recorded on a ShimadzueUV
3600 UVeViseNIR Spectrophotometer, equipped with a Varian
Cary single cell Peltier accessory and fluorescence measurements
were performed with a Varian Cary Elipse Spectrofluorometer
equipped with a multicell cell holder. For the spectrophotometric
titrations we used a Crison pH meter Basic 20þ equipped with
a combined glass electrode, (cod. 50 29), and standardized at 25 ꢀC
using standard buffers of pH 4, 7 and 9.
afford 2 (1.72 g, 75% yield). 1H NMR (CDCl3, 400.15 MHz)
d: 5.19 and
5.20 (2s, 4H, 2xCH2), 7.12 (dt, 1H, J ¼ 8.0 Hz and J ¼ 0.6 Hz, HeAr),
7.23e7.26, 7.32e7.43 and 7.47e7.49 (3m, 12H, HeAr), 10.26 (s, 1H,
CHO). 13C NMR (CDCl3, 100.62 MHz)
d: 71.3 (CH2), 76.5 (CH2), 119.6,
119.9, 124.2, 127.6, 128.3, 128.5, 128.6, 128.7, 128.8, 130.5, 136.3,
151.5, 152.1, 190.2 (CHO); MS (EI) m/z: 318 (M)þ. Anal. Calcd for
C21H18O3 1/4H2O: C, 78.12; H, 5.78; Found: C, 78.46; H, 5.34.
2.2.2. Synthesis of tert-butyl-2,3-dibenzyloxybenzylcarbamate (3)
A solution of 2,3-dibenzyloxybenzaldehyde 2 (1.00 g, 3.14
mmol), t-butylcarbamate (1.11 g, 9.43 mmol), triethylsilane, Et3SiH,
(1.50 mL, 9.43 mmol), trifluoroacetic acid (0.47 mL, 6.30 mmol) in
acetonitrile (40 mL) was stirred at 22 ꢀC for 5 h. The reaction
mixture was precipitated into an aqueous saturated solution of
NaHCO3. The resulting white solid was filtered, washed with water
and dried in vacuum. Compound 3 was obtained as a white solid
(1.26 g) in 93% yield. 1H NMR (CDCl3, 400.15 MHz)
d: 1.42 (s, 9H,
CH3), 4.23 (d, 2H, J ¼ 5.9 Hz, CH2NH), 4.75e4.80 (m, 1H, NH2), 5.07
and 5.14 (2s, 4H, 2xCH2C6H5), 6.90e7.03 (m, 3H, HeAr), 7.31e7.37
(m, 10H, 2xCH2C6H5). 13C NMR (CDCl3, 100.62 MHz)
d: 28.4 (CH3),
40.1 (CH2NH), 70.9 (CH2), 74.9 (CH2), 113.6, 121.7, 124.2, 127.5, 128.0,
128.2, 128.3, 128.5, 128.59, 128.63, 133.3, 136.9, 137.4, 146.2, 151.7,
155.8 (C]O). Anal. Calcd for C26H29NO4 ½CH2Cl2: C, 68.90; N, 3.03;
H, 6.55; Found: C, 68.98; N, 2.67; H, 6.04.
2.2. Synthesis
2.2.1. Synthesis of 2,3-dibenzyloxybenzaldehyde (2)
To
a
solution of 2,3-dihydroxybenzaldehyde
1
(1.00 g,
2.2.3. Synthesis of 2,3-dibenzyloxybenzylamine (4)
7.24 mmol) and K2CO3 (2.20 g, 15.9 mmol) in DMF (20 mL) at 0 ꢀC,
benzyl bromide (1.89 mL, 15.9 mmol) was added drop-by-drop,
A solution of 3 (1.26 g, 3.01 mmol) in trifluoroacetic acid (1.5 mL)
was stirred at 22 ꢀC for 15 min. After that time, the reaction mixture

C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
1449
was neutralized using an aqueous saturated solution of NaHCO3.
The resulting solid was filtered, washed with water, dried and
purified by flash chromatography using chloroform as eluent to
remove a small amount of unchanged starting material and
a mixture of chloroform/methanol (9:1) to obtain compound 4
(ESIeTOF): calcd. for C28H22NO8 (M þ H)þ 500.1345; Found
500.1340.
2.3. Spectrophotometric measurements
(0.73 mg, 78%). 1H NMR (CDCl3, 400.15 MHz)
d
: 3.74 (s, 2H, CH2NH2),
Electronic absorption spectra were recorded using quartz cells
with 1 cm path length.
5.08 and 5.12 (2s, 2H, 2xCH2C6H5), 6.85 (dd, 1H, J ¼ 7.3 and
J ¼ 1.8 Hz, AreH), 6.95 (dd, 1H, J ¼ 8.2 and J ¼ 1.8 Hz, AreH), 6.99
(dd, 1H, J ¼ 8.2 and J ¼ 7.3 Hz, AreH), 7.28e7.46 (m, 10H,
Acidity constants of 2,3-dihydroxybenzaldehyde(1) and Cat1 e
50
m
M each e were obtained in aqueous solution prepared in
2xCH2C6H5). 13C NMR (CDCl3, 100.62 MHz)
d
: 40.3 (CH2NH2), 70.9
double de-ionized water (conductivity less than 0.1 m
S cmꢁ1), by
(CH2), 75.0 (CH2), 113.9, 121.5, 124.4, 127.5, 128.1, 128.3, 128.4, 128.5,
128.60, 128.63, 134.0, 136.7, 137.2, 145.9, 151.7. HRMS-EI: calcd. for
C21H21NO2 (M)þ. 319.1572; Found 319.1564.
dilution of the DMSO stock solution of Cat1 (DMSO percentage in
the final aqueous solution was always kept less than 1% of the total
volume), and with final ionic strength 0.1 M NaCl. After each pH
adjustment, with strong acid or base, the solution was transferred
into the cuvette, and the absorption spectra were recorded. Spectra
were acquired between 225 and 650 nm (1 nm resolution), at 25 ꢀC.
A typical experiment included more than 10 solutions in which, at
least, 10 different pH values were fixed, in a range between 3 and 12
for 1 and 2e13 for Cat1. The pKa values were determined by using
the program pHab 2006 [24] and the associated errors were
determined using the Albert and Sergeant theory [25]. The species
distribution curves were plotted with the HySS 2009 program [26].
2.2.4. Synthesis of conjugate (5)
a) using standard conditions: N,N0-dicyclohexylcarbodiimide
(DCC) (1.081 g, 5.24 mmol) and N-hydroxysuccinimide (0.603 g,
5.24 mmol) were added to a solution of 5(6)-carboxyfluorescein
(1.517 g, 4.03 mmol) in dried DMF (4 mL) were added. The
reaction solution was stirred at room temperature overnight
(16 h), under argon atmosphere, to complete the intermediate
formation of the activated ester. Subsequently, the resulting
white precipitate was filtered off and an amount of 4 (1.545 g,
4.84 mmol) was added to the filtrate. The mixture was stirred at
room temperature for 24 h, followed by solvent removal under
reduced pressure. The resulting residue was taken up in chlo-
roform and the pH was adjusted with a solution of hydrochloric
acid (10%) to pH 3 and extracted with a mixture of chloroform/
methanol (8:2). After dried under high vacuum, the crude
product was purified by chromatography column using
a mixture of chloroform/methanol (8.5:1.5) as eluent. Conjugate
5 (0.180 g) was obtained in 27% yield.
2.4. Fluorescence measurements
Cat1 fluorescence emission measurements were performed in
1 cm cuvettes and all the spectra were recorded at 25.0 ꢂ 0.1 ꢀC,
with excitation and emission slit widths of
5 nm, with
lexc ¼ 493 nm and in the range 495e750 nm.
Stock solutions of Cat1 were obtained by preparing a concen-
trated solution in DMSO. Samples for the pH dependence of the
emission fluorescence intensity study, in a pH range of 2e13, were
prepared by dilution of a known volume of the DMSO stock solution
b) using microwave irradiation: A mixture of 5(6)-carboxy-
fluorescein (97.1 mg, 0.258 mmol), DCC (58.1 mg, 0.282 mmol),
N-hydroxysuccinimide (32.4 mg, 0.282 mmol), 4 (75.0 mg,
0.235 mmol) and dried DMF (2 mL) was placed in a 10 mL
reaction vial, which was then closed under argon atmosphere
and placed in the cavity of a CEM microwave reactor. The
reaction vial was irradiated (1 min ramp to 55 ꢀC and 60 min
hold at 55 ꢀC, using 100 W maximum power). The reaction
work-up was similar to the described before affording 14.5 mg
of conjugate 5 (26% yield).1H NMR (40- and 50-isomers, MeOD,
in water, to a final concentration of 5 mM and final ionic strength
0.1 M NaCl. Solution’s pH was adjusted by adding small aliquots of
strong acid or base and DMSO percentage in the final aqueous
solution was always kept less than 1% of the total volume.
2.5. Fluorescence quenching
Fluorescence quenching measurements were performed in
MOPS buffer (pH 7.4) at 25 ꢀC. Stock solutions of the different metal
ions were acquired [Fe(NO3)3, Al(NO3)3, Cu(NO3)2 and Zn(NO3)2]
from SigmaeAldrich and stabilized with nitrilotriacetic acid triso-
dium salt (NTA) at a 1:5 proportion. Once again Cat1 solution was
prepared by dilution, of a known volume of the DMSO stock solu-
400.15 MHz) d: 4.49 and 4.62 (2s, CH2NH), 5.00, 5.11, 5.13 and
5.18 (4s, CH2C6H5), 6.53 (dd, J 8.8 and J 2.3 Hz, HeAr), 6.60 (d, J
8.8 Hz, HeAr), 6.68 (d, J 2.3 Hz, HeAr), 6.85 (dd, J 8.0 and J
1.6 Hz, HeAr), 6.95e7.57 (m, HeAr), 8.04 (d, J 8.1 Hz, HeAr),
8.08 (dd, J 8.1 and J 1.2 Hz, HeAr), 8.15 (dd, J 8.1 and J 1.2 Hz,
tion, in MOPS buffer to achieve a final 3 mM concentration of Cat1.
DMSO’s percentage in the final aqueous solution was always kept
less than 1% of the total volume. The ligand solution was then
mixed with increasing amounts of the metal stock solution, in
a range of molar ratios from 10:1 to 1:2 of Cat1 to metal ion.
Fluorescence intensities were always corrected for dilution.
The fluorescence quantum yield values for Cat1 and 5(6)-
carboxyfluorescein were determined, at 25 ꢀC, according to the
method of Williams et al. [27] and what is described by Fery-
Forgues and Lavabre [28], using fluorescein as standard [29]. To
minimize reabsorption effects, the absorbance’s sample values
were kept below 0.1 [30].
HeAr), 8.40 (s, HeAr). 13C NMR (MeOD, 100.62 MHz)
d: 40.1
(CH2NH), 72.0, 75.8 and 75.9 (CH2C6H5), 103.6, 114.8, 122.0,
122.2, 125.37, 125.42, 128.9, 129.0, 129.1, 129.3, 129.4, 129.6,
129.7, 130.3, 130.4, 133.3, 133.5, 137.8, 138.50, 138.54, 138.9,
139.0, 147.27, 147.3, 153.2, 153.3, 168.4 (CONH), 170.6 (COOH).
MS (ESI) m/z: 678 (M þ H)þ.
2.2.5. Synthesis of Cat1
A solution of conjugate 5 (228 mg, 0.337 mmol) in methanol
(5 mL) and HCl (0.01 mL) was placed into a hydrogenation vessel.
The air was displaced with N2, a catalytic amount of 10% Pd/C (w/w)
was added and the mixture was stirred at room temperature, with
H2 at 5 bar for 7 h. The reaction mixture was filtered and the solvent
evaporated in vacuum to give the crude product. The resulting
residue was crystallized in methanol/chloroform to give 93.8 mg
(56% yield). 1H NMR see Table 1 in Supporting Information. HRMS
3. Results and discussion
3.1. Synthesis
To synthesise the target fluorescein-based dye functionalized
with a catechol unit (Cat1) we firstly prepared an ortho substituted

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C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
Scheme 2. Synthetic approach to synthesise Cat1.
catechol (1,2-dihydroxybenzene) bearing a methylamine group. The
synthesis is outlined in Scheme 1 and involves (i) benzylation of the
hydroxyl groups of the 2,3-dihydroxybenzaldehyde with benzyl
bromide followed by (ii) condensation with tert-butylcarbamate, in
the presence of triethylsilane and subsequent (iii) hydrolysis in TFA,
using a reductive amination protocol [31]. Using this step’s
sequence, catechol derivative 4 was synthesised in a good yield
(82%).
In order to optimize the conditions to perform the coupling
reaction of 4 with 5(6)-carboxyfluorescein several procedures have
been attempted. Firstly, this reaction was carried out through in situ
generation of the corresponding activated ester using DCC and N-
hydroxysuccinimide as coupling reagents and dried DMF as solvent.
After stirring at room temperature for 40 h, in argon atmosphere,
the reaction provided the desired fluorescein-catechol conjugate 5
in 27% yield. Taking into account the dielectric properties of the
reaction solvent (DMF), we anticipated that microwave irradiation
could be a good alternative process to improve the reaction
performance. In fact, when the same reaction was performed in
only 60 min at 55 ꢀC using a microwave monomode apparatus as
heating source, the expected conjugate 5 was obtained in 26% yield.
This represents a significant reducing of reaction time, from 40 h to
60 min, keeping a similar reaction yield.
Fig. 1) shows (i) one singlet at
singlets at 5.08 and 5.12 ppm from CH2 of the benzyl groups (iii)
three double doublets at 6.85, 6.95 and 6.99 ppm corresponding
to the catechol scaffold protons and (iv) one multiplet at
7.28e7.46 ppm corresponding to the aromatic benzyl group
protons. Once performed the coupling reaction of 4 with the
commercial available 5(6)-carboxyfluorescein, the resulting
conjugate 5 was isolated as a 40- and 50-isomeric mixture. Thus, for
these isomers, the 1H NMR spectrum in methanol-d4 (B, Fig. 1)
revealed to be more complex and the most characteristic signals are
d 3.74 ppm from CH2NH2 (ii) two
d
d
d
(i) a set of two singlets at
CH2NH protons, (ii) a set of four singlets at
5.18 ppm from CH2 of the benzyl groups and (iii) three set of
aromatic signals at 6.52e6.68, 6.84e7.56 and 8.03e8.40 ppm
d
4.49 and 4.62 ppm attributed to the
d
5.00, 5.11, 5.13 and
d
corresponding to fluorescein and catechol scaffolds. Comparing
1
with the H NMR spectra of 40- and 50- isomeric mixture of Cat1,
also in methanol-d4 (C, Fig. 1), the most interesting differences
observed are (i) the absence of the resonance of the benzyl groups
and (ii) the appearing of a group of two singlets at
6.30 ppm attributed to the resonance of H-9 protons.
d 6.19 and
The 1H and 13C NMR signal assignment of Cat1 (available in the
Supporting Information) was achieved by analysis of 1H/1H COSY,
In order to obtain the final compound Cat1, conjugate 5 was
dissolved in
a mixture of methanol/HCl and placed under
a hydrogen atmosphere over 10% Pd/C. Under these conditions,
a yellow powder was isolated which was very soluble in alcoholic
solvents, such as methanol and ethanol, but insoluble in water. Its
NMR spectra, obtained in methanol-d4, are consistent with the Cat1
conjugate (Scheme 2), which results both on the benzyl protecting
groups removal and simultaneous reduction of the double bond at
position 9 of the xanthene ring.
Taking into account previous work with 3,4-HPOs/naphthalene
conjugates [32] this result wasn’t expected under these reaction
conditions. However, a similar result was described in the literature
for a nitro fluorescein derivative using the Raney-Ni reagent as
reduction agent [33]. In order to validate our result, we performed
the same reaction using the starting material 5(6)-carboxy-
fluorescein. A reduction on the double bond in position 9 of fluo-
rescein was achieving, as it was confirmed by the presence of the 9-
H proton at ca. 6.20 ppm correlated with 9-C carbon at ca. 40 ppm,
in NMR spectra. These chemical shift values were in agreement
with the chemical shifts observed for the parent nitro fluorescein
derivative, where proton H-9 appears at 6.15 ppm [33].
3.2. NMR spectroscopy
NMR spectroscopy was of particular importance for the struc-
tural elucidation of the synthesised compounds (see Fig. 1). The 1H
NMR spectrum of the catechol derivative 4 in chloroform-d (A,
1
Fig. 1. H NMR spectra (A) 4, (B) 40- and 50- isomeric mixture of conjugate 5, (C) 40- and
50- isomeric mixture of Cat1.

C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
1451
Fig. 2. (A) UVeVis spectra of 2,3-dihydroxybenzaldehyde in the pH range 2e12. [2,3-dihydroxybenzaldehyde] ¼ 50
m
M, 0.1 M NaCl, 25 ꢀC. (B) Speciation diagram of 2,3-
dihydroxybenzaldehyde as a function of pH.
1H/13C HSQC and 1H/13C HMBC spectra and the determined struc-
tures are presented in Scheme 2. Well separated resonance signals
were observed in all spectra for both isomeric forms (40- isomer and
50 -isomer) and their structures were defined by analysis of the 1H
aldehyde possesses two dissociable protons, corresponding to the
two hydroxyl groups and the values obtained for these two acidity
constants e pKas (see Table 2) are within the usual ranges observed
for catechols [14].
NMR spectrum and the results verified by 1H/1H COSY and 1H/13
C
The UVeVis spectra obtained for 2,3-dihydroxybenzaldehyde in
aqueous solution in the pH range 2e12 are depicted in Fig. 2
together with the corresponding speciation diagram.
The spectra depicted in Fig. 2 show a batochromic displacement
of the 2,3-dihydroxybenzaldehyde bands with increasing pH. The
distribution diagram shows that at physiological pH the species H2L
and (HLꢁ) are present in approximately identical percentage.
According to the NMR characterization, Cat1 possesses 5
protonation sites (H5L, Scheme 3) and the determination of its pKa
values was performed by spectrophotometric titration (the UVeVis
spectra of the pH titration are depicted in Fig. 3).
HMBC spectra. The mole ratio of 40- and 50 -isomers was found to be
1:0.5 as it was determined from the integral intensity of their
characteristic resonance signals in 1H NMR spectrum. The tentative
assignment of the proton and carbon chemical shifts at position 9 in
the molecules was proved on the basis of the multiple bonds
1He13C connectivity’s observed in 1H/13C HMBC spectrum (See
Supporting Information). Well resolved cross peaks due to long
range CH couplings (3JCH) were observed between the proton at
6.30 ppm (H-9) and carbons C-1/8, C-4a/5a, C-1a/8a, C-10, C-60 (in
both forms). Additionally, long range CH couplings (3JCH) were
registered between C-9 (38.7 ppm) and the aromatic protons H-1/8
and H-60 in both isomer forms. The 1H and 13C NMR spectral data
confirm the structure of the two isomeric forms, presented in
Scheme 2.
The spectra shown in Fig. 3 are quite different from those of
Fig. 2 showing a strong band at ca. 500 nm characteristic of the
fluorophore. As pH increases a batochromic displacement of the
band is observed.
The acidity constants values determined for Cat1 (represented
as H5L) are valid under the experimental conditions of this work
and the data refinement was performed assuming the proposed
model Equations (1)e(5).
3.3. Characterization in aqueous solution of 2,3-dihydroxyben-
zaldehyde and Cat1
H5LðaqÞ5H4LꢁðaqÞ þ HþðaqÞ b0;1;ꢁ1;0
H5LðaqÞ5H3L2ꢁðaqÞ þ 2HþðaqÞ b0;1;ꢁ2;0
(1)
(2)
The dissociation constants of catechol, 2,3-dihydroxybenz-
aldehyde, were determined by spectrophotometric titration and
used as comparative term for ligand Cat1. 2,3-Dihydroxybenz-
Scheme 3. Proposed protonation sequence of Cat1.

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C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
Table 2
1.0
0.8
0.6
0.4
0.2
0.0
Acidity constants of 5(6)-carboxyfluorescein, 2,3-dihydroxybenzaldehyde and Cat1.
pKai
5(6)-carboxyfluorescein
[34]
2,3-dihydroxybenzaldehyde
Cat1 (H5L)
pH=13
pKa1
pKa2
pKa3
pKa4
2.08
4.31
6.43
7.97 ꢂ 0.01
4.08 ꢂ 0.06
12.5 ꢂ 0.1
8.2 ꢂ 0.1
12.22 ꢂ 0.03
pH=7
10.4 ꢂ 0.1
The pKa values obtained by UVeVis spectroscopy show that the
pKa values of the hydroxyl groups of C-3 and C-6 are only observed
together, andas amodelin the pHab2006programwehadtoassume
their simultaneous deprotonation thus meaning that the value
calculated for pKa2 accounts for the “sum” of the two pKa values.
Therefore our results allowed the determination of “four” acidity
constants for Cat1, which are in agreement with values determined
for 5(6)-carboxyfluorescein and 2,3-dihydroxybenzaldehyde.
Considering the pKa values determined for Cat1 we plotted the
correspondent species distribution diagram as a function of pH
(Fig. 4). At physiological pH one predominant specie is present,
300.0
400.0
500.0
600.0
λ / nm
Fig. 3. UVeVis spectra of Cat1 in a pH range from 2 to 13 ([Cat1] ¼ 50
mM, 0.1 M NaCl,
25 ꢀC).
[H2L]3ꢁ
.
H5LðaqÞ5H2L3ꢁðaqÞ þ 3HþðaqÞ b0;1;ꢁ3;0
H5LðaqÞ5HL4ꢁðaqÞ þ 4HþðaqÞ b0;1;ꢁ4;0
(3)
(4)
Considering the data obtained (see Table 2) and considering that
at pH above 6.43, fluorescein scaffold should be a dianionic species
[34], and consequently more soluble in water, it can be expected
that be most adequate pH value to study Cat1 in aqueous media is
the physiological one. Considering the protonation scheme
proposed, previous published studies [34] and the results obtained
for Cat1 pKas it should be expected that, at physiological pH, Cat1 is
fully deprotonated in the fluorescein scaffold and protonated at the
catechol unit.
In order to study the behaviour of Cat1 in the presence of metal
ions a profile of the variation of fluorescence intensity with pH was
obtained (Fig. 5). The results show that the fluorescence intensity
increases with the pH reaching a maximum at 7.4.
Considering the results, the potential biological application of
Cat1 and the fluorescent data presented (Fig. 5) which shows that
maximum fluorescence intensity is obtained at pH 7.4 we consid-
ered Cat1 as a good candidate to pursue studies of metal ion
interaction at physiological pH. Before the latter studies the pho-
tophysical characterization of Cat1 and the fluorophores e fluo-
rescein and 5(6)-carboxyfluorescein e was performed at pH 7.4 and
the results namely the lmax for absorption and fluorescence emis-
H5LðaqÞ5L5ꢁðaqÞ þ 5HþðaqÞ b0;1;ꢁ5;0
(5)
The log b0,1,ꢁn,0 determined are presented in Table 1.
According to the proposed model refinement of data allowed
the determination of only four log values. Therefore we tested
b
new set model equations e Eqs. (6)e(9) -, and from their data
refinement we determined the pKa values of Cat1.
H5LðaqÞ5H4LꢁðaqÞ þ HþðaqÞ b0;1;ꢁ1;0
H5LðaqÞ5H2L3ꢁðaqÞ þ 3HþðaqÞ b0;1;ꢁ3;0
(6)
(7)
H5LðaqÞ5HL4ꢁðaqÞ þ 4HþðaqÞ b0;1;ꢁ4;0
H5LðaqÞ5L5ꢁðaqÞ þ 5HþðaqÞ b0;1;ꢁ5;0
(8)
(9)
sion, the molar absorption coefficient (
quantum yield (FF) are shown in Table 3.
3 ) and the fluorescence
The pKa values of catechol units and the fluorescein scaffold [34]
are well documented in literature and are registered in Table 2
together with those determined in this work for 2,3-
dihydroxybenzaldehyde and Cat1.
In fluorescein derivatives the equilibrium between enol and
lactone forms is generallyobserved [23,34]. However in ourcase this
equilibrium is prevented by the presence of the hydrogen atom at C-
9. Taking into account the pKas obtained it is possible to conclude
that: the first pKa1 registered in Table 2 for 5(6)-carboxyfluorescein
does not exist for Cat1. The deprotonation of Cat1 is related to the
hydroxyl groups present in: (i) fluorescein scaffold at carbon atoms
C-3, C-6 and C-20 and (ii) catechol unit at C-200 and C-300.
100
80
60
L
H
L
HL
H
L
H
L
2
4
5
40
20
0
Table 1
Log b0,1,ꢁn,0 values determined for Cat1 considering
the proposed model equations.
log b0,1,ꢁn,0
b0,1,ꢁ1,0
b0,1,ꢁ2,0
b0,1,ꢁ3,0
b0,1,ꢁ4,0
b0,1,ꢁ5,0
ꢁ4.05 ꢂ 0.03
ꢁ10.18 ꢂ 0.06
e
2
4
6
8
10
12
pH
ꢁ25.76 ꢂ 0.07
ꢁ35.76 ꢂ 0.08
Fig. 4. Distribution diagram of Cat1 (50
m
M, 25 ꢀC) as a function of pH

C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
1453
A 800
unquenched
Cat1
600
10:1
400
5:1
200
3:1
2:1-1:2
0
500
525
550
575
600
625
650
λ / nm
Fig. 5. Graphical representation of emission fluorescence intensity percentage of Cat1
B
in a pH range from 2 to 12 (considering 100% of emission fluorescence intensity for pH
7.4).
800
600
400
200
0
The FF values determined for Cat1 and 5(6)-carboxyfluorescein
are lower than the standard fluorophore (fluorescein) as expected
according to their structures.
3.4. Behaviour of Cat1 in the presence of metal ions
The interaction of Cat1 with different metal ions (Fe3þ, Al3þ
,
Zn2þ and Cu2þ) was investigated by examining the variations
observed in the fluorescence intensity of the fluorophore. Evidence
of the corresponding metal complexes formation in solution can be
provided by observation of a significant quenching of fluorescence
intensity. Quenching experiments were therefore performed using
increasing concentrations of the metal ions and a fixed concen-
tration of Cat1. As an example we present in Fig. 6 the emission
fluorescence intensity change of Cat1 with increasing concentra-
tions of Fe(III). The results obtained for the other metal ions tested
are similar to this one but less expressive in terms of the emission
fluorescence intensity decrease.
0.0
2.0x10
4.0x10
6.0x10
3+
|Fe | / M
Fig. 6. (A) Graphical representation of Cat1 (MOPS, pH 7.4, at 25 ꢀC, lexc ¼ 493 nm)
emission fluorescence intensities with increasing Fe(III) concentrations (arrow). Cat1/
metal ratios of 10:1, 5:1, 3:1, 2:1, 1:1, 1:1.6 and 1:2 were tested. (B) Maximum emission
fluorescence intensity (measured at lem ¼ 518 nm) plotted against corresponding
Fe(III) concentrations. Measurements were carried out in triplicate.
From analysis of Fig. 6 we can conclude that the quenching effect
of Fe(III) on Cat1 is almost complete at a ratio 1:3 (metal/ligand) as
expected, considering similar studies with 3,4-HPOs [23] and the
percentage of fluorescence quenching is ca. 70%. For the other metal
ions the decrease in the emission fluorescence intensity is: 19% for
Al3þ, 32% for Cu2þ and 4% for Zn2þ. A comparison of results ob-
77.9%
80
tained for 3 mM solutions of Cat1 together with a 1:1 M ratio of
62.1%
metal ion is shown in Fig. 7.
60
Comparing the obtained data with previous results of an equiv-
alent 3,4-HPO ligand [23], we conclude that the percentage of fluo-
rescence quenching of Cat1 in the presence of Fe(III) is very similar.
Emission fluorescence intensity quenching of Cat1 in the pres-
ence of increasing concentrations of the biologically relevant metal
ions Fe3þ, Al3þ, Zn2þ and Cu2þ is depicted in Fig. 8. The results show
40
17.9%
20
Table 3
Photophysical parameters (lmax for absorption and fluorescence emission,
3 and FF)
3.2%
for Cat1, 5(6)-carboxyfluorescein and fluorescein in MOPS, pH 7.4, at 25 ꢀC.
0
UVeVis
Fluorescence
Fe (III)
Al (III)
Cu (II)
Zn (II)
lmax
3
( ꢃ 105 Mꢁ1
lmax
FF
n+
(nm)
cmꢁ1
)
(nm)
M
Cat1
493
492
490
1.45
5.78
3.10
518
516
510
0.55
0.70
0.78
Fig. 7. Influence of Fe3þ, Al3þ, Cu2þ and Zn2þ on the emission fluorescence intensity of
Cat1. The results were obtained in a 3
M Cat1 solution (10 mM MOPS, pH 7.4, 25 ꢀC)
with a fixed 1:1 M ratio for all the different metal ions understudy.
5(6)-carboxyfluorescein
fluorescein
m

1454
C. Queirós et al. / Dyes and Pigments 93 (2012) 1447e1455
Appendix. Supporting information
1.0
0.8
0.6
0.4
0.2
0.0
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.dyepig.2011.10.010.
Fe(III)
Al(III)
Cu(II))
Zn(II)
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