Sequestering ability to Cu2?+ of a new bodipy-based dye and its behavior as in vitro fluorescent sensor

Article abstract of DOI:10.1016/j.jinorgbio.2016.11.030

A Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) derivative has been conceived and synthesized starting from L-aspartic acid, as a selective turn-off sensor of Cu^2?+ ions. Its acid–base properties were determined to study the formation of

Full text of DOI:10.1016/j.jinorgbio.2016.11.030

Sequestering ability to Cu2 + of a new bodipy-based dye and its behavior as
in vitro fluorescent sensor
Teresa Papalia, Anna Barattucci, Davide Barreca, Ersilia Bellocco, Paola
Bonaccorsi, Lucio Minuti, Marco Sebastiano Nicolo`, Andrea Temperini,
Claudia Foti
PII:
DOI:
Reference:
S0162-0134(16)30479-2
doi: 10.1016/j.jinorgbio.2016.11.030
JIB 10130
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
30 August 2016
23 November 2016
30 November 2016
Please cite this article as: Teresa Papalia, Anna Barattucci, Davide Barreca, Er-
silia Bellocco, Paola Bonaccorsi, Lucio Minuti, Marco Sebastiano Nicol`o, Andrea Tem-
perini, Claudia Foti, Sequestering ability to Cu2 + of a new bodipy-based dye and its
behavior as in vitro fluorescent sensor, Journal of Inorganic Biochemistry (2016), doi:
10.1016/j.jinorgbio.2016.11.030
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ACCEPTED MANUSCRIPT
Sequestering Ability to Cu2+ of a New Bodipy-based Dye and its Behavior
as in vitro Fluorescent Sensor
Teresa Papalia,a† Anna Barattucci,a Davide Barreca,a Ersilia Bellocco,a Paola Bonaccorsi,a*
Lucio Minuti,b Marco Sebastiano Nicolò,a Andrea Temperini,c and Claudia Foti.a*
aDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali - ChiBioFarAm,
Università di Messina, 98166 Messina, Italy
bDipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di Sotto 8,
06123 Perugia, Italy
cDipartimento di Scienze Farmaceutiche, Università di Perugia, Via del Liceo 1, 06123 Perugia,
Italy
† Dipartimento di Scienze agrarie, Università Mediterranea, Feo di Vito, 89124 Reggio Calabria,
Italy
Corresponding authors: Paola Bonaccorsi pbonaccorsi@unime.it; Claudia Foti cfoti@unime.it
Author e-mails:
teresa.papalia@unirc.it
abarattucci@unime.it
dbarreca@unime.it
ebellocco@unime.it
lucio.minuti@unipg.it
mnicolo@unime.it
andrea.temperini@unipg.it

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Abstract
A Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) derivative has been conceived and
synthesized starting from L-aspartic acid, as a selective turn-off sensor of Cu2+ ions. Its acid-
base properties were determined to study the formation of metal/sensor complex species by
titration of solutions each containing a different metal ion, such as Cu2+, Ca2+, Zn2+, Pb2+ and
Hg2+ and different metal/sensor ratios. The speciation models allowed us to simulate the
distribution of the metal/sensor complex species at the normal concentrations of the
corresponding metals present in biological fluids. The distribution diagrams, obtained by varying
the concentration of sensor 1, clearly indicate that sensor 1 responds selectively to Cu2+ at
micromolar concentrations, even in the presence of other more abundant metal cations Ca2+.
Finally, we analyzed the cellular uptake of sensor 1 on human erythrocytes and its ability to
chelate Cu2+ in the cellular environment. Results indicate that it crosses the plasmatic
membrane and colors the cells of a bright fluorescent red. Exposing the fluorescent cells to Cu2+
results in a complete cellular photobleaching of the red fluorescence, indicating that sensor 1 is
able to detect metal changes in the cytosolic environment.
Keywords :
Cu2+ Sensor
Bodipy
Fluorescence
Complexing ability
Cell internalization

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1. Introduction
Although metal ions are necessary for the functioning of all cells in living organisms, changes
in their distribution is one of the main reasons for diseases and aging. For instance, copper is
the third most abundant essential element in biological systems where it plays a vital role in
various physiological processes.[1] However, serious problems to humans can be caused by
either a deficiency or by high levels of this ion: a copper deficiency causes Menke’s disease,
while high concentrations in the liver are known to produce Wilson’s disease.[2] Additionally, it
has been demonstrated that regulation of the concentration of Cu2+ ions plays a role in the
pathogenesis of Alzheimer’s and Parkinson’s diseases,[2] cardiovascular disease,[3] and
diabetes.[4]
Sensing metal ions has become essential, due to their effects on human health. The
widespread use of confocal fluorescence microscopy has driven the development of functional
fluorescent reagents that respond to intracellular events, thereby offering a powerful tool to
study cellular metal ion accumulation, trafficking, or toxicity with a sensitivity comparable to
other traditional methodologies, such as histochemical and radioactivity techniques.[5]
A typical fluorescent chemosensor usually contains a binding moiety as metal receptor and a
fluorophore for signal transduction. However, effective fluorescent probes for imaging metal ions
in living cellular systems have to meet several strict requirements. Most importantly, a sensor
must be selective for a specific metal ion over other biologically abundant cations, including the
most common Na+, K+ and Ca2+. Metal probes also have to be compatible with biological
systems: they must be permeable to the cellular plasmatic membrane, have low or zero toxicity
and their water solubility is also important. Finally, high optical brightness values of the sensor
can reduce the amount needed for cellular application and thus minimize cellular alterations.
Although the challenge is high, the design and synthesis of fluorescent chemosensors with high
selectivity and sensitivity for metal ions remain an exciting field of research for chemists and
biologists.

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Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes have unique properties that
include: relatively high quantum yields, large extinction coefficients, narrow emission band
widths, excitation and emission wavelengths in the visible region, stability to light and chemical
and good solubility.[6] They also offer the possibility of a number of structural changes and
functionalization of their basic scaffold.[7] Significant attention has been devoted to the use of
Bodipy dyes in many research areas such as ion sensing and signaling,[8] fluorescent
switches,[9] light-harvesting systems,[10] and photosensitizers in photodynamic therapy.[11]
In continuing our exploitation of biocompatible luminescent probes,[10,12] we have
conceived and synthesized a new Bodipy-based sensor with six potential sites available for
binding with the metal cation, composed of four carboxyl and two amide groups.[13] Sensor 1,
shown in Fig 1, was synthesized from L-aspartic acid to develop a new fluorescent probe with a
specific selectivity toward Cu2+. It is well known that paramagnetic Cu2+ significantly quenches
fluorescence and thus compound 1 may act as turn-off fluorescent chemosensor. In this paper
the sequestering properties of sensor 1 were evaluated by studying the ability of the molecule
not only to complex Cu2+ but also other divalent metals: specifically two essential, Ca2+ and Zn2+
and two toxic, Pb2+ and Hg2+, metal cations. The aim was to identify the speciation of each
system (i.e. the distribution of the components between the different chemical forms), and,
therefore the distribution, where different metal cations are simultaneously present at
concentrations normally found in biological fluids. Moreover, the cellular uptake of sensor 1 and
its ability to chelate Cu2+ were tested on human erythrocytes, offering an easy and useful cell
model to analyze the plasmatic membrane permeability of molecules.[14]

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Figure 1. Synthetic pathway for Sensor 1.
2. Materials and Methods
2.1 Chemicals
Solvents were purified following standard procedures. All of the reactions were monitored by
TLC on commercially available precoated plates (silica gel 60 F254), and the products were

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visualized with vanillin [1 g dissolved in MeOH (60 mL) and conc. H2SO4(0.6 mL)] and under UV
lamp. Silica gel 60 was used for column chromatography. Solutions of metal ions were prepared
by weighing the corresponding salts, CuCl2H2O, CaCl2, ZnCl2, Pb(NO3)2, HgCl2 (Fluka, Sigma
Aldrich or Riedel-de-Haen), and were standardized with EDTA standard solutions. Their purity
was always ≥ 99 %. Concentrated ampoules of hydrochloric acid and sodium hydroxide (Fluka)
were used to prepare the corresponding diluted solutions. The hydrochloric acid solutions were
standardized against sodium carbonate, those of sodium hydroxide against potassium hydrogen
phthalate. The aqueous solution of sensor 1 was prepared by pre-dissolving a weighed amount
in a very small volume of ethanol. All solutions were protected from atmospheric CO2 using
soda lime traps. Grade A glassware and twice-distilled water were employed in the preparation
of all solutions.
2.2 Synthesis
Proton (1H) and carbon (13C) NMR spectra were recorded on a Varian 500 spectrometer (at
500 MHz for 1H; and125 MHz for 13C) using CDCl3 as solvent, unless otherwise stated.
Chemical shifts are given in parts per million (ppm) ( relative to residual solvent peak for 1H
and 13C), coupling constants (J) are given in hertz, and the attributions are supported by
Heteronuclear Single Quantum Coherence (HSQC) experiments and COrrelation SpectroscopY
(COSY). Elemental analysis were determined by a FISON EA1108 and are expressed in %.
The syntheses of L-Aspartic acid diethylester 4 and aldehyde 5 are reported in Supplementary
content (SI).
General procedure for the Knoevenagel condensation of aldehyde 5 with Bodipy 6.
Piperidine (140 l) and few crystals of p-TSOH were added to a solution of Bodipy 6 (50 mg,
0,11 mmol) and aldehyde 5 (0,22 mmol) in Toluene (8 ml), using a round bottom flask equipped
with a Dean-Stark apparatus. The reaction was monitored by Tlc (Toluene/EtOAc 90:10). The
solution was refluxed for 48 h. Solvent was removed under reduced pressure. The reaction
mixture was purified by column chromatography (Toluene/EtOAc up to 85:15).

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Bodipy 7. This was obtained as first product from the column chromatography (Toluene/EtOAc
90:10). The orange-colored fractions were collected and the solvent was removed under
pressure to give a fuchsia solid (7 mg, 30%). Rf 0,431H NMR (CDCl3) δ 7.87 (d, 2H, Ar-I), 7.82
(d, 2H, Ar-C=O), 7.73 (AB system, 1H, JA-B= 16.6 Hz, HA-C=CH), 7.65 (d, 2H, Ar-C=O), 7.21
(AB system, 1H, HB-C=CH), 7.09 (d, 2H, Ar-I), 6.64 (s, 1H, Py), 6.06 (s, 1H, Py), 5.07-5.05
(broad m, 1H, CH), 4.30-4.26 (m, 2H, CH2CH3), 4.19-4.17 (m, 2H, CH2CH3), 3.14 (AB system,
1H, JA-B= 17.6 Hz, HA-CH), 2.90 (AB system, 1H, HB-CH), 2.62 (s, 3H, CH3), 1.49 (s, 3H, CH3),
1.46 (s, 3H, CH3), 1.32-1.26 (m, 6H, 2 x CH2CH3). 13C NMR: 171.1, 170.7, 166.3, 157.2,
149,9, 139.9, 138.3, 134.4, 133.3, 132.8, 130.0, 128.2, 127.6, 127.4, 121.8, 121.0, 117.7, 94.8,
62.0, 61.1, 49.0, 36.3, 29.6, 25.3, 14.8, 14.7, 14.1, 14.0.Anal. Calcd for C35H35BF2IN3O5
(753,38): C 55.80, H 4.68, N 5.58. Found: C 56.02, H 4.69, N 5.6.
Bodipy 8. This was obtained as second product from the column chromatography
(Toluene/EtOAc 85:15). The blue colored fractions were collected and the solvent was removed
under pressure to give a deep blue solid (17 mg, 45%). It was also obtained (70%) from
condensation of Bodipy 7 (1 eq.) with aldehyde 5 (1 eq.), following the general procedure. Rf
0,13 1H NMR (CDCl3):7.89 (d, 2H, Ar-I), 7.86 (d, 4H, 2 x Ar-C=O), 7.80 (AB system, 2H, JA-B
=
16.7 Hz, 2 x HA-C=CH), 7.70 (d, 4H, 2 x Ar-C=O), 7.28 (AB system, 2H, 2 x HB-C=CH), 7.12 (d,
2H, Ar-I), 6.69 (s, 2H, Py), 5.07-5.05 (broad m, 2H, CH), 4.30-4.26 (m, 4H, 2 x CH2CH3), 4.21-
4.16 (m, 4H, 2 x CH2CH3), 3.15 (AB system, 2H, JA-B = 17.6 Hz, 2 x HA-CH), 2.99 (AB system,
2H, 2 x HB-CH), 1.51 (s, 6H, 2 x CH3), 1.33-1.26 (m, 12H, 4 x CH2CH3).13C NMR: 171.1,
170.7, 166.3, 152.5, 142.3, 139.8, 138.4, 138.1, 135.2, 134.4, 133.5, 130.2, 127.7, 127.6,
120.9, 118.4, 94.9, 62.0, 61.1, 49.1, 49.0, 36.3, 29.6, 29.2, 14.9, 14.1.Calcd for C51H52BF2IN4O10
(1056,69): C 57.97, H 4.96, N 5.30. Found: C 58.04, H 4.97, N 5.31.
Sensor 1. Bodipy 8 (13 mg, 0,012 mmol) was dissolved in DCM/MeOH (1:1, 12 ml). The blue
solution was cooled at 0°C and a 2M solution of KOH in MeOH (2,6 g in 6 ml, 0,048 mmol) was
added dropwise. The reaction mixture was stirred for 1 h and HCl 2N (20 ml) was added. The
dark green solution was extracted with DCM (3 x 10 ml), made anhydrous, and solvent was
removed under reduced pressure. The solid was washed twice with DCM and twice with

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Toluene (9 mg, 80%). 1H NMR (Acetone-D):  8.09 (d, 2H, 2 x NH), 8.04 (d, 2H, Ar-I), 8.02 (d,
4H, 2 x Ar-C=O), 7.81 (AB system, 2H, JA-B= 16.1 Hz, 2 x HA-C=CH), 7.77 (d, 4H, 2 x Ar-C=O),
7.61 (AB system, 2H, 2 x HB-C=CH), 7.36 (d, 2H, Ar-I), 6.97 (s, 2H, Py), 5.07-5.03 (m, 2H, 2 x
CH), 3.04-3.03 (m, 4H, 2 x CH2), 1.55 (s, 6H, 2 x CH3).13C NMR: 173.1, 173.0, 167.3, 154.1,
144.1, 141.1, 140.3, 140.0, 137.2, 135.9, 134.8, 132.2, 129.6, 128.7, 121.9, 120.1, 96.3, 51.0,
50.9, 37.1, 31.0, 15.7.Calcd for C43H36BF2IN4O10 (944,48): C 54.68, H 3.84, N 5.93. Found: C
54.62, H 3.83, N 5.92.
2.3 Spectrophotometric measurements
The spectrophotometric measurements of aqueous solutions were recorded, in the range of
300 to 800 nm, using a Varian Cary 50 UV–vis spectrophotometer equipped with an optic fibre
probe having a fixed 1 cm path length. The spectrophotometer was connected to a PC for the
acquisition of the experimental data (absorbance vs wavelength), by Varian Cary WinUV
(version 3.00) software. Simultaneously, in the thermostated measurement cell, the pH of
solution was measured by a combined glass electrode (Ross type 8102, from Thermo/Orion)
connected to a Metrohm 713 potentiometer. The combined glass electrode was standardized
before each experiment on the pH scale. Thereby, simultaneously plotting Absorbance vs 
(nm) and pH vs volume of titrant (mL). The titrant was delivered in the measurement cell by
means of a 665 Metrohm automatic burette; the solutions were vigorously stirred during titration.
The acid-base behavior of sensor 1 was assessed adopting a procedure previously tested on
other systems[15]: 20 mL of solutions containing sensor 1 (L) (0.05 ≤ CL ≤ 0.3 mM) and HCl (0
≤ CHCl ≤ 1 mM) were analyzed varying the pH through alkalimetric titration. For the
investigations on metal complex species, 20 mL of solutions containing different amounts of
sensor 1 (0.005  C  0.04 mM), HCl (0 ≤ CHCl ≤ 2 mM) and metal (0.005 ≤ CM ≤ 0.01 mM) were
analyzed in the pH range 2.5 ≤ pH ≤ 8.

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2.4 Erythrocytes isolation
Donors volunteers participating in the study were informed of all risks, discomforts and
benefits relating to the study, in accordance with the Declaration of Helsinki for research
protocols approved by the institutional review boards of the National Institutes of Health (NIH).
All subjects provided medical histories, using a standardized questionnaire and declared they
had not taken anti-inflammatory medication or nutritional supplements. Blood was obtained by
venipuncture from healthy male volunteers and collected in heparinized tubes. Erythrocytes
were separated from plasma and buffy coat and washed three times with l0 volume of 0.9 %
NaCl and centrifuged at 2,500 rpm for 5 min. During the last washing the packed cells were
resuspended in the incubation buffer (phosphate saline buffer), at pH 7.4 and utilized for
subsequent experiments.
2.5 Cellular internalization and cytotoxic effects
Erythrocytes were incubated for 2 hours at 37°C in the absence or in the presence of the
following final concentrations of sensor 1 of 50, 25, 10, 5, 1, 0,5, 0,1 0.05 and 0.0025 µM. At the
end of incubation time, cells were centrifuged at 2,500 rpm for 5 min. The supernatant was
analyzed to detect the presence of haemoglobin and release of lactate dehydrogenase, while
the pocked cells were resuspended and washed three times in incubation buffer and finally
used for fluorescence and visible microscopy. The supernatant was utilized to check cytotoxicity
by measuring hemolysis and lactate dehydrogenase (LDH) release from damaged cells into
culture medium. The hemolysis of erythrocytes was determined spectrophotometrically at 576
nm based on the ratio of Hb released from cells and expressed as fold of control. LDH activity in
the medium was determined using a commercially available kit from BioSystems S.A. The
concentrations of sensor 1 used in the experiment, did not interfere with LDH determination. For
fluorescence and visible microscopy 10 μL of each samples was placed on a glass slide
covered with a coverslip and monitored using a Leica DMRE microscope equipped with a
fluorescence attachment. TRITC, filter was used for sensor 1 excitation and emission.

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2.6 Bleaching effect of the Cu2+ on sensor 1 internalized in Erytrhocytes.
To test the chelating activity of sensor 1 on Cu2+ ions, erythrocytes were incubated, as above
described, with 10 M of sensor 1 for 2 h, to which 50 µM of Cu2SO4 was added to the reaction
mixtures and left for a further 2 h. Blank, without tested molecules, has been prepared and
incubated in the same experimental conditions. At the end of the incubation time, 10 μL of each
samples were placed on a glass slide covered with a coverslip and monitored by means of a
Leica DMRE microscope equipped with a fluorescence attachment. TRITC, filter was used for
sensor 1 excitation and emission. Images were acquired by a camera connected to the
microscope.
2.7 Statistical analysis
Data are presented as means ± standard deviation (S.D.). Data were analyzed by one-way
analysis of variance (ANOVA).The significance of the difference from the respective controls for
each experimental test condition was assayed by using Tukey’s for each paired experiment. A
P<0.05 was regarded as indicating a significant difference.
3. Results and Discussion
3.1 Synthesis
L-Aspartic acid, possessing two carboxyl groups, appeared an interesting starting point for
the synthesis of a sensor for copper ion. Few other examples are reported in the literature.[16]
The introduction of two molecules of L-aspartic acid onto the terminal part of a Bodipy scaffold
through the formation of two amide bonds would transform the fluorescent probe into a sort of
nipper in which up to six binding sites are represented by the four carboxyl groups and the two
amides, the latter binding to the copper ion through the carbonyl oxygen.[17] Amide donors are
of considerable importance in biology, where they act as coordinating groupsfor metal ions.[18]

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Preparation of precursors 5 and 6 was carried out following well-known procedures.
Commercially available 4-formylbenzoic acid 2 was transformed into the acyl chloride 3, which
was reacted with the diethyl ester of L-aspartic acid 4 to obtain suitably substituted aldehyde 5
in good yields (Fig. 1). The diester 4 was obtained from L-aspartic acid, according to the
synthetic route reported in SI.[19] Bodipy 6 was synthesized following a one pot procedure.[20]
Knoevenagel condensation of aldehyde 5[21] with Bodipy 6 was conducted in the presence of
piperidine as base and a catalytic amount of p-TsOH and afforded, despite different conditions,
always yielded a mixture of the monosubstituted Bodipy 7 and the desired Bodipy 8 (75%
overall yield), these being easily separated and purified by column chromatography. The
recovered Bodipy 7 was converted into Bodipy 8 by condensation with aldehyde 5, under
conditions previously described, with a 70% yield. This repetitive step was necessary to
increase the yield of Bodipy 8, considering as longer reaction times or drastic conditions in the
first condensation led to lower yields of the 7+8 mixture and not an increment of compound 8.
Finally, hydrolysis of Bodipy 8 gave sensor 1 in 80% yield (Fig.1). Sensor 1 was purified by
washing with toluene and DCM several times. The structure of sensor 1 was fully characterized
by analysis of its 1H NMR, 13C NMR,HSQC and COSY spectra (see SI).
0.3
pH = 2.34
pH = 3.56
pH = 5.77
pH = 6.31
pH = 6.63
0.2
0.1
0.0
pH = 7.83
pH = 8.19
300
400
500
600
700
800
 (nm)
Figure 2. Example of spectrophotometric titration on a 0.01 mM solution of sensor 1, at I = 0.15
M and t = 37°C.

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3.2 Speciation studies
The possibility of using sensor 1 (L) for the determination of Cu2+ in biological fluids was
evaluated through the determination of its ability to complex not only Cu2+, but also cations of
two essential metals, Ca2+ and Zn2+ and two toxic, Pb2+ and Hg2+, metal cations. This was done
in order to ascertain the speciation of each systems individually (i.e. the distribution of the
components between the different chemical forms) and, therefore, to simulate the distribution in
the simultaneous presence of different metal cations at the concentrations normally found in
biological fluids.
Given that the study of metal-ligand interactions requires the knowledge of the acid-base
behavior of individual components, the first step of the study concerned the determination of the
molecule protonation constants. Data reported in Table 1 were employed for metal hydrolysis.
[22]
Table 1. Metal hydrolysis constant values employed
a)
p,q
logp,q
Ca2+
Cu2+
Zn2+
Pb2+
Hg2+
-3.40
-6.07
-21.1
1 1
1 2
1 3
1 4
2 1
2 2
2 6
3 4
4 4
6 6
-13.05
-8.22
-8.94
-17.1
-28.4
-40.7
-9.0
-7.55
-17.03
-17.53
-27.80
-39.12
-42.9
-2.7
-10.16
-57.5
-27.5
-21.8
-19.6
a) values calculated at I = 0.15 M from ref [22] and referred to the reaction: p M2+ + q H2O =
Mp(OH)q + q H+

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Table 2. Protonation and complex formation constants for sensor 1 - metal systems, at I = 0.15
M and t = 37 °C
Reactiona), b)
log
H+ + L = HL
7.67 ±0.03c)
11.27 ±0.08
13.58 ±0.07
2 H+ + L = H2L
3 H+ + L = H3L
Cu + L = CuL
9.96 ±0.03
18.76 ±0.04
18.66 ±0.01
Cu + H + L = CuHL
2 Cu + L = Cu2L
Ca+ L = CaL
Zn + L = ZnL
9.47±0.08
6.06±0.09
Pb + L = PbL
6.74 ±0.04
12.26 ±0.02
12.53 ±0.02
10.29 ±0.06
Pb + H + L = PbHL
2 Pb + L = Pb2L
Pb + 2L = PbL2
Hg + L = HgL
12.83 ±0.02
15.06 ±0.02
Hg + H + L = HgHL
a) L = sensor 1; b) charges omitted for simplicity; c) ±3 std.dev.

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An example of spectrophotometric titration conducted on a 0.01 mM solution of sensor 1, in
the pH range 2  pH  8 is reported in Fig. 2. Other examples of spectrophotometric titrations of
sensor 1 in the presence of metals are reported in Supplementary Information. Data, obtained
by the procedure outlined in the experimental section, were processed using a Hyspec
program.[23] In the evaluation of complex formation constants, the spectral range of 300 ≤  ≤
700 nm was taken into consideration. The protonation constants obtained under experimental
conditions described are reported in Table 2. The distribution is presented in Fig. 3a showing
that the monoproptonated HL species prevails in a broader pH range (4 ≤ pH ≤ 7.), while H2L
reaches a maximum formation percentage (70%) at pH = 3, and the H3L species at pH < 2.
However, more than 50% of the totally deprotonated L species are only obtained at pH > 7.5 to
obtain.
Having ascertained the acid-base properties of the sensor and the individual metals, it was
possible to determine the formation of complex species by titration of solutions containing
different metal/ligand ratios, as described in the experimental section above. The processing of
spectrophotometric data revealed the formation of complex species reported in Table 2,
together with the corresponding formation constant values.
Cu2+ forms three different complex species: CuL, CuLH and the dinuclear Cu2L. The species
distribution vs pH is shown in Fig. 3b. Under the experimental conditions, Cu2+ is present as
monoprotonated CuHL species in the range 2 ≤ pH ≤ 6; at pH > 6 the dinuclear Cu2L prevails,
while the formation of CuL is negligible. It is only between pH 4 and 5 that 20% of metal is
present as the free metal cation Cu2+. At blood pH values (pH = 7.4), 20% of metal cation is
present as CuLH, and slightly less than 80% as Cu2L.

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100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
CaL
LH
Ca
CuLH
Cu L
2
LH
2
LH
3
L
Cu
CuL
8
2
4
6
8
2
4
6
2
4
6
8
pH
pH
pH
(a)
(b)
(c)
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
HgL
HgH
-
2
Zn
Pb
Pb L
2
PbL
ZnL
HgLH
Hg
PbLH
ZnH
-
1
2
4
6
8
2
4
6
8
2
4
6
8
pH
pH
pH
(d)
(e)
(f)
Figure 3. Distribution diagram vs. pH of H+, Cu2+, Ca2+, Zn2+, Pb2+ and Hg2+ species, at I =0.15
M and t = 37 °C. Experimental conditions: CM = CL = 10 M (L = sensor 1).
The simplest systems are obtained with Ca2+ or Zn2+, which lead to the formation of single
ML complexes, as can be observed from the distribution diagrams (Fig. 3c-3d). Most of Ca2+ is
present in this form for pH > 5. The stability of ZnL species is of two orders of magnitude lower
than that of Ca2+ (logK = 9.64 and 6.06 for CaL and ZnL, respectively). This is reflected in the
species distribution and, in the 2  pH  6 interval, Zn2+ is present as a free ion and the
formation of ZnL becomes prevalent for pH> 7.
The speciation model of the Pb2+ system appears more complex, with the formation of PbL,
PbLH, Pb2L and PbL2 species (Fig. 3e). The distribution diagram shows that for pH < 5, most of
Pb2+ is present as a free ion, with a maximum of 20% formation of the PbLH species at pH = 4.

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The Pb2L and PbL species reach 50% formation at pH = 6.5 and pH = 8, respectively. Under
these conditions, the formation of PbL2 species is negligible.
The last metal cation Hg2+ forms HgL and HgHL species, the formation of which is significant
for pH < 6 (Fig. 3f). At blood pH, Hg2+ is present almost exclusively as hydrolytic Hg(OH)2
species.
100
80
60
40
20
0
100
80
60
40
20
0
CaL
Cu L
CuLH
2
LH
3
LH
2
CaL
8
CuLH
Cu L
2
2
4
6
8
2
4
6
pH
pH
(a)
(b)
Figure 4. Distribution diagrams vs pH in multicomponent solution. L = sensor 1.
Experimental conditions: CCu = 18M, CCa = 200M, CZn = 20M, CPb = 1 nM and CHg = 1 nM
(a) CSens = 1 mM; (b) CL = 1 M.
3.3 Complexing ability in biological systems
Having determined the speciation of individual systems, it was possible to simulate their
distribution in the presence of the different metal cations at the concentrations normally present
in biological fluids. For this purpose, concentrations normally present in blood were considered
for Cu2+, Ca2+ and Zn2+ (CCu = 18M, CCa = 200M, CZn = 20M),[24] while for the two toxic
metals (Pb2+ and Hg2+) nanomolar concentrations were evaluated (CPb = 1 nM and CHg = 1 nM)
since they should not normally be present in a biological fluids.
Distribution diagrams obtained for fixed concentrations of the metals and varying the
concentration of sensor 1 are reported in Fig. 4. At relatively high concentrations of sensor

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(Csens = 1 mM, Figure 3a), over pH = 3, the diagram shows a >95% formation rate of CaL
species . Although the stability of the CaL is of the same order of magnitude as CuL (logK =
9.96 and 9.47 for CuL and CaL respectively), the much higher concentration of Ca2+ in the blood
(CCa = 200 M) determines the distribution shown in Fig. 4.
Decreasing sensor 1 concentration, increases the percentage of molecule present as Cu2+
complex and decreases the formation rate of the Ca2+ complex. In particular, when the sensor
concentration is in the micromolar order, all Cu2+ is complexed by sensor 1 at blood pH and the
formation of other species is negligible. Under these conditions sensor 1 responds selectively to
Cu2+, even in the presence of the other metal cations.
B
A
C
E
F
D
Figure 5. Fluorescence microscopy images of erythrocytes incubated with 10 (A), 5 (B), 1 (C),
0.5 (D), 0.1 (E) and 0.05 (F) µM (final concentrations) of sensor 1.
3.4 Cellular internalization studies

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Cellular internalization of sensor 1 was analyzed by fluorescence microscopy after 2 h of
erythrocyte incubation in the absence or in the presence of different amounts of the tested
compound. In the range of concentration of 0.05-10 µM sensor 1 enters the cells and illuminates
them with a red fluorescence (Fig. 5).
5.0
A
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
**
**
a
b
c
d
e
f
g
h
i
l
6
5
4
3
2
1
0
B
**
**
a
b
c
d
e
f
g
h
i
l
Figure 6. Spectroscopic determination of LDH (A) and hemoglobin (B) release from
erythrocytes incubated for 2 h in the absence (a) or in the presence of 50 (b), 25 (c), 10 (d), 5
(e), 1 (f), 0.5 (g), 0.1 (h), 0.05 (i) or 0.025 (l) µM of sensor 1. Samples were analyzed by one-
way ANOVA, followed by Turkey’s test. Asterisks (**) indicate a significant difference with
respect to controls (P<0.05).
Fluorescence microscopy images disclose a clear fluorescence of the cytosol, supporting
that sensor 1 is able to cross the plasmatic membrane. Moreover fluorescence microscopy

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highlights points with high concentration of the dye, probably attributable to the strong binding of
the bodipy to proteins.
Figure 7. Fluorescence and light microscope images of erythrocytes incubated with sensor 1 in
the absence (A and C, respectively) or in the presence (B and D, respectively) of CuSO4.
Erythrocytes were first incubated with 10 M of sensor 1 and after 50 µM of Cu2SO4 were added
to the reaction mixtures. At the end of the incubation time, 10 μL of each samples were placed
on a glass slide covered with a coverslip and monitored by means of a microscope.
Erythrocyte internalization is evident even at low concentrations of dye (0.1-0.05 M),
indicating how the tested compound can be useful for biological purposes, even at

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concentrations lower or comparable to those used for other fluorescent dyes, this reducing any
potential toxic effects. In fact, hemolysis and LDH release analysis clearly showed that high
sensor 1 concentrations (50 and 25 M) induced cell damage, while lower concentrations did
not produce any significant change on the cell structure compared to control, as shown in Fig.
6. The results obtained for speciation and chelating activity by UV-Visible spectroscopy led us to
further analyze the ability of sensor 1 to form chelates with Cu2+ in the biological system studied.
Therefore, we first incubated cells with 10 M of sensor 1 (a concentration at which we found a
good fluorescence response with no evident toxicity effects), then added the metal to the
incubated cells and, finally, incubated them for a further 2h. The results are depicted in Fig. 7.
Samples without Cu2+ shows a clear red fluorescence emission, while the ones incubated with
Cu2+ are almost completely dark.
4. Conclusions
In conclusion, a new BODIPY-based dye, which emits in the red region, was prepared and
characterized, and its ability to complex Cu2+ investigated, together with its cell internalization in
human erythrocytes. Speciation studies indicated, at blood pH (pH = 7.4), the most abundant
species to be the one where two equivalents of Cu2+ are complexed with sensor 1. Furthermore,
a study on the complexing properties conducted on a number of cations showed that sensor 1
responds selectively to Cu2+ at micromolar concentrations. The intense fluorescence of this dye
allowed us to study its cell internalization by fluorescence microscopy, using concentrations in
micro and nanomolar ranges. At these concentrations, compound 1 acts as a Cu2+ turn-off
chemosensor in human erythrocytes exposed to Cu2+ solution without compromising their
cellular viability. Finally, the structural characteristics of compound 1 leave open the possibility
of further substituent changes that could improve its promising properties; therefore it appears
to be a lead compound as a metal probe in biological systems.

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This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Pictogram for Graphycal abstract
Cu2+
100
0.3
pH = 2.34
CuLH
Cu
L
pH = 3.56
pH = 5.77
pH = 6.31
pH = 6.63
pH = 7.83
pH = 8.19
2
80
60
40
20
0
0.2
0.1
0.0
CaL
8
300
400
500
600
700
800
2
4
6
pH
 (nm)
Synopsis for the Graphical abstract
The synthesis of a new Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based
chemosensor was developed. The sensor shows selective sequestering properties towards
Cu2+ at micromolar concentrations. It crosses membrane of erythrocytes, colors them with a
red fluorescence and is sensible to Cu2+ variation in cells.

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Highlights
•
A Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based chemosensor was
synthesis
•
Sensor shows selective sequestering properties towards Cu2+ at micromolar
concentrations
•
•
The sensor crosses membrane of erythrocytes and colors them with a red fluorescence
The sensor is sensible to Cu2+ variation in the erythrocytes
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