Design of a water soluble 1,8-naphthalimide/3-hydroxy-4-pyridinone conjugate: Investigation of its spectroscopic properties at variable pH and in the presence of Fe3+, Cu2+ and Zn2+

Article abstract of DOI:10.1016/j.dyepig.2013.02.020

The synthesis and sensing properties of a new fluorescent probe designed to have a 4-amino-1,8-naphthalimide fluorescent platform functionalized with a 3-hydroxy-4-pyridinone bidentate chelating unit at the 4-position and a terminal aliphatic dimethylamino group at the imide site, are reported. The absorption and fluorescence properties of the ligand were investigated in DMSO and in aqueous solution at variable pH and in the presence of increasing concentration of Fe³⁺, Cu²⁺ and Zn²⁺. Analysis of the UV-Vis spectra at variable pH allowed the determination of three pK_a values (pK_a1 = 3.19, pK_a2 = 8.38, pK_a3 = 9.95) and establishment of the corresponding speciation diagram. Fluorescence spectra obtained in the same conditions show that the fluorescence intensity of the probe decreases with increasing pH and are off above pH 9 as a result of photo-induced electron transfer arising from the aliphatic dimethylamino group. Under physiological pH conditions, the probe shows an absorption band centred at 439 nm and emits in the green at λ = 536 nm. Analysis of UV-Vis and EPR spectra of the ligand in the presence of Fe³⁺ and Cu²⁺ is consistent with the formation of the corresponding metal ion complexes. The fluorescence intensity of the ligand is quenched in the presence of variable concentrations of Fe³⁺, Cu²⁺ and Zn²⁺ and under physiological pH conditions the fluorescence of the probe is ca 92%, 88% and 91% quenched in the presence of Fe³⁺, Cu²⁺ and Zn ²⁺ respectively.

Full text of DOI:10.1016/j.dyepig.2013.02.020

Accepted Manuscript
Design of a water soluble 1,8-naphthalimide/3-hydroxy-4-pyridinone conjugate:
investigation of its spectroscopic properties at variable pH and in the presence of
Fe3+, Cu2+ and Zn2+
Tânia Moniz, Carla Queirós, Rita Ferreira, Andreia Leite, Paula Gameiro, Ana M.G.
Silva, Maria Rangel
PII:
S0143-7208(13)00070-3
10.1016/j.dyepig.2013.02.020
DYPI 3867
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 9 January 2013
Revised Date: 22 February 2013
Accepted Date: 25 February 2013
Please cite this article as: Moniz, T, Queirós, C, Ferreira, R, Leite, A, Gameiro, P, Silva, AMG, Rangel
M, Design of a water soluble 1,8-naphthalimide/3-hydroxy-4-pyridinone conjugate: investigation of
its spectroscopic properties at variable pH and in the presence of Fe3+, Cu2+and Zn2+, Dyes and
Pigments (2013), doi: 10.1016/j.dyepig.2013.02.020.
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ACCEPTED MANUSCRIPT
Design of a water soluble 1,8-naphthalimide/3-hydroxy-4-pyridinone conjugate: investigation of its
spectroscopic properties at variable pH and in the presence of Fe3+, Cu2+ and Zn2+
Tânia Moniz, Carla Queirós, Rita Ferreira, Andreia Leite, Paula Gameiro, Ana M.G. Silva and Maria Rangel

ACCEPTED MANUSCRIPT
Design of a water soluble 1,8-naphthalimide/3-hydroxy-4-pyridinone conjugate: investigation of
its spectroscopic properties at variable pH and in the presence of Fe3+, Cu2+ and Zn2+
Tânia Moniz,a Carla Queirós,a Rita Ferreira,a Andreia Leite,a Paula Gameiro,a Ana M.G. Silva,a* and
Maria Rangelb*
a
REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-
007 Porto, Portugal
b REQUIMTE, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4099-003 Porto, Portugal
*mcrangel@fc.up.pt; ana.silva@fc.up.pt
Abstract
The synthesis and sensing properties of a new fluorescent probe designed to have a 4-amino-
1,8-naphthalimide fluorescent platform functionalized with a 3-hydroxy-4-pyridinone
bidentate chelating unit at the 4-position and a terminal aliphatic dimethylamino group at the
imide site, are reported. The absorption and fluorescence properties of the ligand were
investigated in DMSO and in aqueous solution at variable pH and in the presence of
increasing concentration of Fe3+, Cu2+ and Zn2+.
Analysis of the UV-Vis spectra at variable pH allowed the determination of three pKa values
(pKa1 =3.19, pKa2 = 8.38, pKa3 = 9.95) and establishment of the corresponding speciation
diagram. Fluorescence spectra obtained in the same conditions show that the fluorescence
intensity of the probe decreases with increasing pH and are off above pH 9 as a result of
photo-induced electron transfer arising from the aliphatic dimethylamino group. Under
physiological pH conditions, the probe shows an absorption band centred at 439 nm and emits
in the green at λ= 536 nm.
Analysis of UV-Vis and EPR spectra of the ligand in the presence of Fe3+ and Cu2+ is
consistent with the formation of the corresponding metal ion complexes. The fluorescence
intensity of the ligand is quenched in the presence of variable concentrations of Fe3+, Cu2+ and
Zn2+ and under physiological pH conditions the fluorescence of the probe is ca 92%, 88% and
91% quenched in the presence of Fe3+, Cu2+ and Zn2+ respectively.
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Keywords
4-amino-1,8-naphthalimides, 3-hydroxy-4-pyridinones, microwave-assisted synthesis,
fluorescence properties
Introduction
Since the early reports by Tsien and co-workers regarding fluorescent probes for calcium,1,2
the design of highly efficient fluorescent probes for sensing and monitoring various chemical
species has received an increasing interest from the scientific and industrial communities.
Indeed, many fluorescent probes have been developed for different applications including
probes sensitive to pH,3 to metal ions,4 to monitor enzymatic activity,5 reactive oxygen and
nitrogen species (ROS and RNS)6 and many other analytes.7
The most general design concept of fluorescent metal ion probes involves the use of the
photoinduced electron transfer (PET) model based on the covalent linkage of a single or
branched chelating architecture and a fluorophore unit. The covalent linkage which separates
the two units is typically a short aliphatic spacer that minimizes any ground-state interactions.
In these probes, the interaction with the analyte will cause significant changes in the emission
spectra accompanied by no, or minor, changes in the absorption spectra of the probes.8
Originally detailed by de Silva et al.9a,b and Czarnik et al.,9c,d this process has been widely
used in the construction of many fluorescent probes, with special emphasis for 4-amino-1,8-
naphthalimide based fluorescent probes.10
4-Amino-1,8-naphthalimides are fluorophores that exhibit bands with an ICT (intramolecular
charge transfer) character caused by the electronic conjugation of the electron donating amine
and the electron withdrawing imide (Fig. 1).11 The ICT character gives rise to a large excited-
state dipole and broad absorption and emission bands typically centred at ca 450 and 550 nm,
respectively, when recorded in water.10 The spectroscopic properties of 4-amino-1,8-
naphthalimides are also highly solvent dependent, noting that polar protic solvents stabilize
the ICT character more than apolar solvents. The compounds have fairly simple structures for
which facile and straightforward synthetic methods have been established and their structure
allows changes on either the amine function in the 4-position of the aromatic naphthalene
moiety, or at the imide nitrogen atom.12 Consequently, the 4-amino-1,8-naphthalimides have
been extensively used as strongly absorbing and colorful fluorophores in the design of
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fluorescent probes, particularly for sensing biologically relevant metal ions, such as Cu2+ and
Zn2+,13 and heavy metal ions, like Hg2+ and Pd2+.14
In most examples, 4-bromo-1,8-naphthalimide, which is a very weak fluorescent compound,
has been used as precursor to reach a variety of metal sensing fluorescent probes based on the
4-amino-1,8-naphthalimide platform. These fluorescent probes have been synthesized using
mainly two synthetic strategies: (i) direct nucleophilic displacement of the bromine atom of
the 4-bromo-1,8-naphthalimide with a functional amine or (ii) initial reaction with a linker
and subsequent introduction of the functional moiety. These synthetic approaches have been
chosen for the design of many metal sensing fluorescent probes where, in most instances, the
nitrogen atom introduced in position 4 of the naphthalimide molecule can be involved in
metal ion coordination.15
Hydroxypyridinones are an important class of N-heterocyclic bidentate chelators which show
high affinity towards several biological important metal ions, such as Fe(III).16 This type of
chelator, which include analogues of 1,2-hydroxypyridinone (1,2-HPO), 2,3-
hydroxypyridinone (2,3-HPO) and 3,4- hydroxypyridinone (3,4-HPO), have been widely used
as decorporation agents for the medical treatment of iron overload associated with several
diseases.17 Also some hydroxypyridonate complexes have been applied as Fe(III) chelators
for reducing iron toxicity.18
Among the different types of hydroxypyridinones, the 3,4-HPO class is particularly attractive
for pharmaceutical purposes since their structure allows tailoring of their
hydrophilic/lipophilic balance (HLB) without significantly changing its chelating properties.19
Variations in HLB can be achieved by simply introducing appropriate substituents on the
endocyclic nitrogen atom of the pyridinone ring, the 3-hydroxy-4-pyridinones are synthesized
by the reaction of 3-hydroxy-4-pyrones with primary amines, thus leading to the optimal
lipophilicity for delivery or removal of metal ions in the body.19
In recent years, many 3,4-HPOs modified with fluorescent platforms, like coumarin or
fluorescein, have been developed by the Hider´s laboratory. The latter probes have shown to
be selective fluorescence quenchers for Fe3+ up to 90% and found application in the
determination of cellular iron concentrations.20 Lately, Callan et al. summarized the
development of various molecular and supramolecular fluorescent sensors that have been
successfully used for detection of Fe3+, many of them based on 3,4-HPO derivatives.21
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Our group has long been interested in the synthesis and solution properties of 3,4-HPO
ligands for biomedical applications19,22 and more recently on the design of metal ion
sensors.23 Lately, we prepared two bidentate ligands bearing a 3,4-HPO chelator moiety and a
naphthalene fluorescent platform that can be used to sense metal ions in organic solvents but
due to their very low solubility and lack of fluorescent properties in water are not usable to
monitor metal ions in aqueous media.23a
In the present work we report the design and properties of a new 3,4-HPO probe with high
solubility in water and suitable fluorescence properties in aqueous solution. In order to
achieve our purpose and in view of the well-known properties of 1,8-naphthalimide
derivatives we coupled a 3,4-HPO bidentate chelating unit with the 4-amino-1,8-
naphthalimide platform (Fig. 2). The chelator backbone is connected to the fluorophore
through the ortho position relative to one of the chelating oxygen atoms of the 3,4-HPO
moiety, thus providing a more rigid structure and a close proximity between the fluorophore
and the binding unit. The 1,8-naphthalimide platform was modified at the imide nitrogen
atom in order to accommodate a terminal aliphatic amino group that increases solubility in a
pH range determined by the pKa of the amino group. To the best of our knowledge this is the
first report of a molecule conjugating the 1,8-naphthalimide platform with a 3-hydroxy-4-
pyridinone chelator.
Experimental
Materials and instrumentation
Chemicals were obtained from Sigma–Aldrich (grade puriss, p.a.) and were used as received
unless otherwise specified.
Nuclear magnetic resonance spectra (1H and 13C NMR) were measured on a Bruker III
1
13
Avance 400 spectrometer operating at 400.15 MHz and 100.62 MHz for H and C spectra,
respectively. Chemical shifts (δ) are reported in parts per million and coupling constants (J) in
1
hertz; internal standard was TMS. Unequivocal H assignments were made with aid of 2D
gCOSY (1H/1H), while 13C assignments were made on the basis of 2D gHSQC (1H/13C) and
gHMBC experiments (delay for long range J C/H couplings were optimized for 7 Hz). Mass
spectrometry analysis were performed at the University of Santiago de Compostela (Spain).
Melting points were measured in a glass capillary tube on a Stuart Scientific SMP1 apparatus
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and are uncorrected. FTIR spectra were obtained with a Perkin Elmer FT-IR System
(Spectrum BX) with ATR (Attenuated Total Reflectance).
Synthesis
Synthesis of N-(dimethylamino)ethyl-4-bromo-1,8-naphthalimide (3)
Following reported procedures,40 a mixture of 4-bromo-1,8-naphthalic anhydride 1
(0.10 g, 0.36 mmol), N,N-dimethylethylenediamine 2 (0.05 mL, 0.43 mmol) and ethanol (1
mL) was heated under reflux for 4 hours. After cooling to room temperature (RT), the solid
that formed was filtered, washed with water and dried to yield quantitatively naphthalimide 3.
1H NMR (400 MHz, CDCl3) δ: 2.35 (s, 6H, N(CH3)2CH2CH2-), 2.65 (t, 2H, J 7.2 Hz,
N(CH3)2CH2CH2-), 4.32 (t, 2H, J 7.2 Hz, N(CH3)2CH2CH2-), 7.85 (dd, 1H, J 8.4 and J 7.2
Hz, H-6), 8.05 (d, 1H, J 7.8 Hz, H-3), 8.42 (d, 1H, J 7.8 Hz, H-2), 8.58 (d, 1H, J 8.4 and J 0.8
Hz, H-5 or H-7), 8.66 (d, 1H, J 7.2 and J 0.8 Hz, H-5 or H-7).
Synthesis of N-(dimethylamino)ethyl-4-(2'-aminomethyl-1',6'-dimethyl-3'-benxyloxy-4'-
pyridinone)-1,8-naphthalimide (5)
The procedure to synthesize the compound was adapted from those described in the
literature.41 A pressure vial of 10 mL was charged with naphthalimide 3 (0.130 g, 0.375
24
mmol), 1,6-dimethyl-2-aminomethyl-3-benzyloxy-4-pyridinone 4
(0.194 g, 0.75 mmol),
Pd(OAc)2 (4.2 mg, 0.019 mmol), BINAP (11.8 mg, 0.019 mmol), t-BuOK (42.1 mg, 0.375
mmol) and DMF (0.5 mL) and the cap was tightened thoroughly. The vessel was exposed to
microwave irradiation at 130ºC (1 min ramp to 130ºC and 4 min hold at 130ºC, using a
maximum power of 150 W). The reaction tube was thereafter cooled to RT and the mixture
was purified by flash chromatography and preparative TLC using a mixture of
chloroform/methanol (9:1). Product 5 was crystallized in chloroform/methanol to give 126.3
mg (64% of yield) of a yellow powder. Mp 205-207 ºC. IR (cm-1) 2956 (N-H), 1682, 1646,
1622 (C=O), 1582, 1576, 1554, 1532, 1506, 1456, 1392, 1364, 1326, 1290, 1244, 1222, 1142,
1120, 1098, 1048, 1028, 972, 954, 842, 774, 746, 698. 1H NMR (400 MHz, CDCl3) δ: 2.33 (s,
3H, 6’-CH3), 2.40 (s, 6H, N(CH3)2CH2CH2-), 2.70 (t, 2H, J 7.2 Hz, N(CH3)2CH2CH2-), 3.56
(s, 3H, 1’-CH3), 4.27 (d, 2H, J 4.0 Hz, HNCH2), 4.34 (t, 2H, J 7.2 Hz, N(CH3)2CH2CH2-),
5.10 (s, 2H, CH2C6H5), 6.29 (s, 1H, H-5’), 6.53 (d, 1H, J 8.4 Hz, H-3), 7.00-7.03 and 7.10-
7.13 (2m, 5H, CH2C6H5), 7.61 (dd, 1H, J 8.3 and J 7.4 Hz, H-6), 8.42 (d, 1H, J 8.3Hz, H-2),
13
8.51 (d, 1H, J 8.3 Hz, H-5 or H-7), 8.56 (d, 1H, J 7.4 Hz, H-5 or H-7). C NMR (100 MHz,
5

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CDCl3) δ: 20.9 (6’-CH3), 36.2 (1’-CH3), 37.8 (CH2CH2N(CH3)2), 39.7 (HNCH2), 45.7
(N(CH3)2CH2CH2-), 57.0 (N(CH3)2CH2CH2-), 73.1 (CH2C6H5), 104.5 (C-3), 111.4 (C-1a),
118.8 (C-5’), 121.2 (C-4a), 122.7 (C-7b), 124.9 (C-6), 128.09 (C-7), 128.2, 128.4, 129.6,
129.7, 131.3, 134.2, 136.6, 139.6 (C-2’), 146.0 (C-3’), 147.9 (C-6’), 149.3, 164.1 (C-1), 164.7
(C-8), 173.4 (C-4’). MS (FAB) m/z: 525 [M+H]+. HRMS (FAB) m/z Calcd. for C31H33N4O4
525.2502; Found 525.2501.
Synthesis of N-(dimethylamino)ethyl-4-(2'-aminomethyl-1',6'-dimethyl-3'-hydroxy-4'-
pyridinone)-1,8-naphthalimide dihydrochloride (N-3,4-HPO)
A 1 M solution of boron trichloride in dichloromethane (1.5 mL) was dropped slowly
into an ice-bath-cooled suspension of derivative 5 (106.1 mg, 0.202 mmol) in dry
dichloromethane (10 mL), under an argon atmosphere. The mixture was stirred at room
temperature for 5 h. Methanol (5 mL) was added to quench the reaction. After removal of the
solvent in vacuum, the residue was precipitated with methanol/ acetone to afford the
hydrochloride salt of N-3,4-HPO (102.6 mg, 73% of yield) isolated as an yellow powder. Mp
213-215 ºC. IR (cm-1) 3310 (O-H), 2906 (N-H), 1682, 1634, 1616 (C=O), 1586, 1576, 1538,
1504, 1480, 1392, 1354, 1280, 1248, 1204, 1120, 1102, 1036, 1020, 960, 940, 898, 784, 754,
1
586. H NMR (400 MHz, DMSO-d6) δ: 2.57 (s, 3H, 6’-CH3), 2.87 (d, 6H, J 4.0 Hz,
+
+NH(CH3)2CH2CH2-), 3.40-3.42 (m, 2H, NH(CH3)2CH2CH2-), 3.97 (s, 3H, 1’-CH3), 4.35-
4.36 (m, 2H, +NH(CH3)2CH2CH2-), 4.91 (d, 2H, J 3.6 Hz, CH2NH), 7.01 (d, 1H, J 8.6 Hz, H-
3), 7.30 (s, 1H, H-5’), 7.73 (dd, 1H, J 8.4 and J 7.4 Hz, H-6), 8.31 (d, 1H, J 8.6 Hz, H-2), 8.48
(d, 1H, J 7.4 Hz, H-7), 8.75 (br. s, 2H, OH), 8.92 (d, 1H, J 8.4 Hz, H-5) 9.97 (br. s, 1H,
+NH(CH3)2CH2CH2-). 13C NMR (100 MHz, DMSO-d6) δ: 20.8 (6’-CH3), 34.8
(+NH(CH3)2CH2CH2-), 38.5 (HNCH2), 40.0 (1’-CH3, under DMSO signal), 42.6
(+NH(CH3)2CH2CH2-), 54.8 (+NH(CH3)2CH2CH2-), 104.4 (C-3), 109.2, 112.7 (C-5’), 120.5
(C-7b), 122.0, 124.8 (C-6), 128.5, 129.4 (C-5), 130.9 (C-7), 134.1 (C-2), 139.1 (C-2’), 143.2
1
(C-3’), 148.6 (C-6’), 149.1, 150.0 (C-4), 160.0 (C-4’), 163.3 (C-1), 164.3 (C-8). H NMR
+
(400 MHz, D2O+DCl) δ: 2.55 (s, 3H, 6’-CH3), 2.94 (s, 6H, NH(CH3)2CH2CH2-), 3.33 (m,
+
+
2H, NH(CH3)2CH2CH2-), 3.98 (s, 3H, 1’-CH3), 4.18 (m, 2H, NH(CH3)2CH2CH2-), 4.80 (s,
2H, CH2NH), 6.52 (m, 1H, H-3), 7.12 (s, 1H, H-5’), 7.32 (m, 1H, H-6), 7.69 (m, 1H, H-2),
7.91 and 8.01 (2m, 2H, H-5 and H-7). 13C NMR (100 MHz, D2O+DCl) δ: 21.4 (6’-CH3), 36.0
(+NH(CH3)2CH2CH2-), 39.4 (HNCH2), 40.0 (1’-CH3), 44.3 (+NH(CH3)2CH2CH2-), 56.4
(+NH(CH3)2CH2CH2-), 105.6 (C-3), 108.8, 114.1 (C-5’), 120.5, 120.9, 125.7 (C-6), 129.3 (C-
5 or C-7), 129.7, 132.2 (C-5 or C-7), 135.3 (C-2), 139.1, 143.8 (C-3’), 151.2, 151.6, 160.0 (C-
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4’), 165.2 (C-1 or C-8), 166.1 (C-1 or C-8). MS (ESI) m/z 435 [M+H]+. HRMS (ESI) m/z
Calcd. for C24H27N4O4 435.2044; Found 435.2027.
Absorption and Fluorescence spectroscopic measurements
Absorption spectra were acquired with a Perkin Elmer Lambda 25 spectrophotometer
equipped with a constant-temperature cell holder, at 25ºC, in 1 cm cuvettes, in the wavelength
range 225-650 nm.
Fluorescence measurements were performed in a Varian Cary Eclipse fluorometer, equipped
with a constant-temperature cell holder, at 25ºC, in 1 cm cuvettes.
Stock solutions of the different compounds were obtained by preparing a concentrated
solution of the compound in dimethylsulfoxide (DMSO). Samples for absorption and
fluorescence measurements were prepared by dilution of the appropriate volume of the
DMSO stock solution. The percentage of the DMSO stock solution in the final volume of the
MOPS buffer solutions was always less than 1%.
Quantum yield determination
The fluorescence quantum yield values for 1,8-naphthalic anhydride (1) and N-3,4-HPO and
5(6)-carboxyfluorescein were determined, at 25ºC, according to the method of Williams et
al30 and what is described by Fery-Forgues and Lavabre,42 using fluorescein as standard.31 To
minimize reabsorption effects, the absorbance’s sample values were kept below 0.1.
Absorption and fluorescence spectra of N-3,4-HPO at variable pH
The characterization in aqueous solution was performed at 25ºC and at variable pH. For
variable pH measurements we started from a solution prepared as previously described and
aliquots of strong acid or base were added to adjust pH to the desired value. After each pH
adjustment the solution was transferred into the cuvette, and the absorption spectra were
recorded. Spectra were acquired at 25°C and between 225 and 800 nm (1 nm resolution).
For the spectroscopic data the pH values were measured with a Crison pH meter Basic 20+
which is equipped with a combined glass electrode, and standardized at 25°C using standard
buffers of pH 4, 7 and 9. The UV-Vis spectra obtained to determine pKa values were
recorded, at each pH, using a Varian Cary bio50 double beam spectrophotometer, equipped
with a Varian Cary single cell Peltier accessory controlled by a computer. The pKa values
were calculated using the program HypSpec.34 The errors reported in this work were
7

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calculated by the method suggested by Albert and Sarjeant.43 The distribution diagrams were
plotted with the program HySS 2008.44
Fluorescence spectra at variable pH were obtained using several buffers and considering the
concentration requirements for fluorescence spectroscopy.
Evaluation of the interaction of the probe N-3,4-HPO with Fe3+, Cu2+ and Zn2+
The evaluation of the interaction of probe N-3,4-HPO with metal ions was performed in
DMSO and in MOPS buffer (pH 7.4, I=0.1 M NaCl) at 25ºC. Stock solutions of the different
metal ions were acquired [Fe(NO3)3, Cu(NO3)2 and Zn(NO3)2] from Sigma-Aldrich and
stabilized with nitrilotriacetic acid trisodium salt (NTA) at a 1:5 proportion. Concentrated
solutions of N-3,4-HPO in DMSO were prepared and used as stock solutions. To prepare the
solution for fluorescence measurements, a known volume of N-3,4-HPO was diluted with
MOPS buffer to achieve a final concentration of, approximately, 3 µM. Increasing amounts of
the metal stock solutions were added to the 3 µM solution of the probe N-3,4-HPO, in a range
of molar ratios from 1:0.01 to 1:1 (chelator:metal ion).
The UV-Vis spectra were recorded after each addition using a Varian Cary bio50 double
beam spectrophotometer, equipped with a Varian Cary single cell Peltier accessory controlled
by a computer. Fluorescence measurements were performed using a Varian Cary Eclipse
fluorometer. To minimize reabsorption effects, only solutions with absorbance below 0.1
were used and fluorescence measurements were obtained above the excitation wavelength.
Fluorescence intensities were always corrected for dilution.
EPR spectra were recorded in DMSO at room temperature and in frozen solution at 100K
using an X-band (9 GHz) Bruker ELEXYS spectrometer equipped with a variable
temperature unit. The samples were prepared by dissolution of the compound in dried DMSO
and placed in a capilary which was placed in a quartz tube.
Results and Discussion
Synthesis and characterization of 1,8-naphthalimide-modified 3,4-HPO (N-3,4-HPO)
The precursor of the chemosensing ensemble, 4-bromo-1,8-naphthalimide 3 (Scheme 1), was
synthesized by condensation of 1,8-naphthalic anhydride 1 with an excess of N,N-
dimethylethylenediamine 2, left under reflux in ethanol for 4 hours. Then, in order to prepare
8

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the protected form of ligand N-3,4-HPO (derivative 5, Scheme 1), the Buchwald–Hartwig
reaction was carried out by adding 4-bromo-1,8-naphthalimide 3 and 1,6-dimethyl-2-
aminomethyl-3-benzyloxy-4-pyridinone 4,24 in the presence of Pd(OAc)2 as precatalyst, 2,2’-
bis(diphenylphosphanyl)-1,1’-binaphthyl (BINAP) as ligand and t-BuOK as base, in DMF,
using conventional heating procedure (oil bath, 4 hours of reflux). After evaporation of the
solvent, the reaction components were purified by preparative TLC using a mixture of
chloroform/methanol (9:1) and characterized by NMR. Following this protocol, derivative 5
was obtained in only 10% of yield (approximately 80% of the starting naphthalimide was
recovered) while a by-product was also isolated. This by-product was identified as
corresponding to N-(dimethylamino)ethyl-4-dimethylamino-1,8-naphthalimide resulting from
the reaction of 4-bromo-1,8-naphthalimide 3 with dimethylamine. In fact, DMF has been used
as source of dimethylamine in a big number of substitution reactions and it has been
speculated that the formation of dimethylamine arises from the decomposition of DMF and
happens when the solvent is heated during long periods.25,26 In order to confirm this behaviour
of DMF, we carried out the reaction of 4-bromo-1,8-naphthalimide 3 in DMF reflux for ca. 10
hours, and obtained exclusively N-(dimethylamino)ethyl-4-dimethylamino-1,8-naphthalimide
in 10% yield (approximately 90% of the starting naphthalimide was recovered unchanged).
In order to improve the reaction outcome to synthesize 5, to the detriment of the synthesis of
N-(dimethylamino)ethyl-4-dimethylamino-1,8-naphthalimide, some modifications were
performed on the synthetic procedure. Microwave-assisted synthesis was used as an
alternative to the traditional oil bath in order to obtain the desired compound 5 in good yield
and the results showed that the reaction time was considerably shorter thus indicating that an
improved synthetic protocol is possible. Therefore, in a monomode reactor, under closed-
vessel conditions (130ºC, 4 min), the reaction of 3 and a 2-fold excess of 4 was performed in
the presence of Pd(OAc)2, BINAP and t-BuOK, in DMF. After chromatographic purification,
the yield of formation of derivative 5 was significantly improved to 64%. This result shows
that, since the reaction time was significantly reduced to 4 min, the degradation of DMF was
minimized and, consequently, the side-reaction of dimethylamine resulting from DMF
degradation was no longer observed.
The benzyl protecting group of 5 was removed with BCl3 in dichloromethane, under argon
atmosphere, affording the expected dihydrochloride salt of ligand N-3,4-HPO in 73% of
yield.
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NMR spectroscopy
The ligand N-3,4-HPO was isolated as +2 charged dihydrochloride salt; one charge is located
in the nitrogen atom of the dimethylethylenediamine arm and the other is placed in the
nitrogen atom of the dihydroxypyridinium residue.
1
This is elucidated in the H NMR spectrum performed in DMSO-d6 by the resonance of the
+
following signals: (i) the multiplet at 3.40-3.42 ppm, corresponding to NH(CH3)2CH2CH2;
(ii) the doublet at 2.87 ppm, corresponding to +NH(CH3)2CH2CH2 and (iii) the broad singlet at
+
9.97 ppm assigned to NH(CH3)2CH2CH2. The assignments of the 13C NMR spectrum have
been achieved on the basis of HSQC and HMBC experiments. The resonance peaks at 42.6
and 54.8 ppm were assigned to +NH(CH3)2CH2CH2 and +NH(CH3)2CH2CH2 and these values
are consistent with the presence of the charge in the nitrogen atom of the
dimethylethylenediamine arm. On the other hand, the singlet corresponding to the resonance
of H-5’ proton, undergoes a downfield shift to 6.97 ppm, as well as the doublet corresponding
to CH2NH protons, which undergo a downfield shift to 5.02 ppm. Both H-5’ and CH2NH
showed strong HMBC correlation with a signal at 143.2 ppm assigned as C-3’ (C-OH). Also
H-5’ showed HMBC correlation with a signal at 160.0 ppm assigned to C-4’ (C-OH). These
values are in close agreement with chemical shifts values found for other (3,4-HPO)s,
confirming that the compound was isolated in the dihydroxypyridinium form.23a
Similar results were obtained when D2O was used as solvent; in this case a few drops of DCl
were added to the NMR tube in order to completely dissolve the compound in D2O. Under
these conditions +NH(CH3)2CH2CH2 and +NH(CH3)2CH2CH2 appear at 44.3 and 56.4 ppm, C-
3’ at 143.8 ppm and C-4’ at 160.0 ppm, confirming the presence of the +2 charged
dihydrochloride salt.
All recorded spectra are provided in supplementary information available.
Electronic spectroscopy
The absorption and fluorescence properties of N-3,4-HPO were studied in the UV-Visible
region and compared with those of its precursors, 1,8-naphthalic anhydride (1) and 4-bromo-
1,8-naphthalimide (3) (formulae in Scheme 1). The spectra obtained in DMSO are shown in
Figures 3a and 3b and those obtained in MOPS buffer solution in Figures 4a and 4b. The
compounds are all soluble in DMSO and concentrated solutions in DMSO can be successfully
dissolved in buffer solution to be further used in cellular studies. As illustrated in Figure 3a
compounds 1 and 3 exhibit quite similar spectra and show bands at 340 and 343 nm,
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characteristic of the π → π* transitions of the π systems in the naphthalene rings. The
similarity of the two spectra is indicative that the conversion of the 4-bromo-1,8-naphthalic
anhydride in 4-bromo-1,8-naphthalimide did not significantly changed the charge distribution
in the fluorophore.
The spectrum of N-3,4-HPO exhibits two sets of bands, one in the range of 281-290 nm,
which corresponds to π → π* transitions of the π system of the 3,4-HPO ligand and the other
band centred at 437 nm which corresponds to π → π* transitions of the π systems of the
naphthalimide fluorophore. The replacement of the bromine group in position 4 of the
fluorophore by an amino group or a derivative significantly changes the charge distribution
through an ICT mechanism. The spectra obtained in a buffer solution at pH 7.4 (Figure 4a)
are very similar to those in DMSO with a slight deviation in the values of λmax as expected for
a different solvent. The data obtained is in agreement with the values reported in the literature
for similar compounds.27-29 The values of λmax and of the molar extinction coefficients of the
three compounds are registered in Table 1. The values obtained are close to those described in
literature for similar compounds27-29 and the usual 10 fold decrease in the value of the molar
extinction coefficient that results from the introduction of the amino group in position 4 of the
naphthalimide fluorophore is also observed for the new compound N-3,4-HPO.
Analysis of the fluorescence spectra of compounds 1, 3, and N-3,4-HPO, depicted in Figures
3b and 4b, shows that compounds 1 and 3 are not fluorescent in DMSO while compound N-
3,4-HPO exhibits an emission band centred at ca 536 nm which is indicative of the
modification of the fluorescent properties of compound 3 upon replacement of the bromine
atom by the amino group of the 3,4-HPO residue. In buffer solution at pH=7.4 (Fig. 4b) we
observed that 1,8-naphthalic anhydride (1) is not fluorescent at all and compound 3 exhibits a
very weak emission at 411 nm. Compound N-3,4-HPO exhibits an emission band centred at
ca 536 nm.
Considering the potential application of the new compound in biological media the
measurements of fluorescence quantum yield (φF) were performed in NaOH 0.1 M and MOPS
buffer (I=0.1M NaCl, pH 7.4) at 25ºC using fluorescein as standard.30 The results are
registered in Table 2 and we observed that for compound N-3,4-HPO an hypochromic
displacement occurs when the pH is lowered to 7.4, thus indicating the sensitivity of the probe
to pH variation in that range. Also, the results illustrate that at pH=7.4 the value of the
fluorescence quantum yield in MOPS buffer is higher than that in NaOH.
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Speciation and evaluation of fluorescence properties of N-3,4-HPO at variable pH
Considering the building molecular fragments of N-3,4-HPO and that the compound is
isolated in the form of a dichloride salt, it is expected that the compound suffers deprotonation
upon dissolution in water. Absorption and fluorescence properties of N-3,4-HPO in aqueous
solution were studied at variable pH in the range 1.5-10.5. The absorption spectra obtained
are displayed in Figure 5 and its analysis show that more than one species is present as pH is
increased and that the spectral peaks are shifted in wavelength and exhibit variable intensity.
Absorption spectra show a decrease in the maximum of absorbance and a bathochromic shift
of the ICT band with the increase of pH. The result is similar to other 4-amino-1,8-
naphthalimide containing pH dependent units.32,33 The analysis of the data collected with the
program HypSpec34 is consistent with the presence of four species and allowed the
determination of three pKa values which are registered in Table 3.
Considering the values of acidity constants reported for 3,4-HPO derivatives, which do not
significantly change with the substituents of the heterocyclic ring and are centred at pKa1~3.5
and pKa2~9.3,35 and those of tertiary amines (pKa~10),36 the formulae of the four species in
which N-3,4-HPO may be present are indicated in Scheme 2. The corresponding speciation
diagram is depicted in Figure 6. The diagram is indicative that at physiological pH conditions
the species H2L+ is predominant and a smaller amount of the neutral form is also present.
Fluorescence spectra of N-3,4-HPO obtained in the range 4 < pH < 10 are displayed in Figure
7. The spectra clearly show that the fluorescence intensity diminishes as pH increases and is
quenched at above pH equal to 9. From the speciation diagram it can be perceived that at pH
9 the neutral species HL, in which the terminal aliphatic amino group is deprotonated, is the
predominant one.
The fluorescence quenching observed at pH > 9 is attributed to a PET process resulting from
deprotonation of the amino group (Scheme 3). The terminal aliphatic amino group attached to
the 4-amino-1,8-naphthalimide platform acts as a quencher and a photo induced electron
transfer process occurs from the lone electron pair of the amino group to the acceptor 1,8-
naphthalimide, making the probe weakly fluorescent.
At physiological pH conditions the fluorescent species H2L+ is predominant and a smaller
amount of the non-fluorescent neutral form is also present.
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The protonation of the amino group diminishes the PET effect and leads to restoration of the
fluorescence of the 4-amino-1,8-naphthalimide fluorophore at lower pH values. Hence, a
marked increase in emission intensity was observed at low pH.37
Interaction of N-3,4-HPO with Fe3+, Cu2+ and Zn2+
Compound N-3,4-HPO includes a 3,4-HPO bidentate unit and consequently formation of
complexes of type FeL3, CuL2 and ZnL2 is expected.16 The affinity of 3,4-HPO ligands
towards M3+ and M2+ is well documented in the literature and the stability constants (log β3
and log β2) are high and in the range 35.0 < log β3 < 37.0 for FeL3 complexes, 17.2 < log β2 <
19.0 for CuL2 complexes and 13.2 < log β2 < 13.7 for ZnL2 complexes.16,22d Also, it has been
shown that the values of protonation constants of 3,4-HPO and the corresponding values of
stability constants for a particular metal ion do not differ significantly with alteration of the
substituents on the pyridinone ring.16
Evidence of the interaction of compound N-3,4-HPO with Fe3+ was assessed, in DMSO and
MOPS buffer, by inspection of the electronic spectra of the ligand in the presence of variable
concentration of Fe3+ and for Cu2+ by analysis of the corresponding EPR spectra at RT and
100K obtained also in DMSO.
In order to judge the use of the new compound N-3,4-HPO to sense Fe3+, Cu2+ and Zn2+, the
interaction of N-3,4-HPO with Fe3+, Cu2+ and Zn2+ was also investigated by examining the
variations observed in the fluorescence spectra of solutions of the probe in MOPS buffer upon
the addition of increasing amounts of the three metal ions.
The absorption and emission spectra of compound N-3,4-HPO for a 3:1 ligand:iron(III) ratio
obtained in DMSO, are shown in in Figures 8a and 8b, respectively. The absorption spectra
clearly show a shift in the ICT band characteristic of the naphthalimide fluorophore and the
appearance of a new broad and much less intense band centred at ca 550 nm characteristic of
formation of an iron(III) complex. Spectra obtained for 2:1 and 1:1 ligand : iron(III) ratios are
provided as supplementary information (Figure S1 in supplementary information). The
fluorescence spectra obtained for the same solutions are indicative that the fluorescence
quenching occurs in the presence of Fe3+ and that the effect is concentration dependent.
Considering that compound N-3,4-HPO is isolated in the form of a dichloride salt and that the
NMR spectrum in DMSO confirms that the amino group in the imide position of the
naphthalimide fluorophore is protonated, we conclude that fluorescence quenching is the
13

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result of metal ion coordination to the 3,4-HPO unit, since in DMSO the deprotonation of the
amino group does not occur and consequently the PET effect observed in aqueous solution is
not active.
The absorption and emission spectra of a solution of N-3,4-HPO in MOPS buffer, upon
addition of increasing amounts of Fe3+ are depicted in (Figures 9a and 9b). In figure 9a it is
clearly shown that as the concentration of Fe3+ is raised the intensity of the bands at ca 320-
370 nm and the ICT absorption band at λmax=439 nm decreases. Also, the maximum of the
ICT band is shifted to longer wavelength and a less intense band emerges at ca 525 nm. These
spectral changes are a consequence of coordination of the pyridinone unit to Fe3+ and are
typical of naphthalimide based probes for sensing cations and anions.38,39 In Figure 9b the
fluorescence spectra of a solution of N-3,4-HPO in MOPS buffer upon addition of increasing
amounts of Fe3+are displayed The spectra show that the intensity of the fluorescence is
diminished as the concentration of Fe3+ is raised and the quenching of fluorescence is ca 92%
at a metal:ligand ratio of 1:3. The quenching is assigned to the formation of the tris iron(III)
complex with the ligand N-3,4-HPO (Scheme 4). In the presence of Cu2+ and Zn2+ the
fluorescence of the ligand is also quenched is ca 88% for Cu2+ and ca 91% for Zn2+ at a
metal:ligand ratio of 1:2. (Figures S2 and S3 in supplementary information)
Confirmation of the coordination of Cu2+ with ligand N-3,4-HPO is provided by the EPR
spectra obtained in DMSO at RT and 100K and shown in (Figures 10a (RT) and 10b (100K)).
The spectrum at RT is well resolved and characteristic of one unpaired electron system,
S=1/2, interacting with a copper nucleus (I= 3/2). The spectrum obtained at 100K is typical of
a copper (II) complex with a rhombic distorted geometry. EPR spectra of copper(II)
complexes with N-alkyl-3,4-HPOs are generally characterized by two g values and
characteristic of square planar complexes with slight distortions in the equatorial plane.23b In
the case of ligand N-3,4-HPO the spectrum shows a higher degree of anisotropy thus
indicating the lower symmetry of the complex which is induced by the nature of the ligand.
Figure 11 shows the structure of the copper(II) complex of ligand N-3,4-HPO obtained with
®
the
software
ChemBio3D
Ultra
(http://www.cambridgesoft.com/Ensemble_for_Chemistry/ChemBio3D/Default.aspx), after
energy minimization. The structure clearly illustrates the distortion induced by the
functionalization of the 3,4-HPO unit with the naphthalimide fluorophore.
14

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Conclusions
The conjugation of a 1,8-naphthalimide platform with a 3-hydroxy-4-pyridinone bidentate
ligand gave rise to a new fluorescent probe, N-3,4-HPO, which is very soluble in water. The
compound exhibits pH dependent spectroscopic properties that allowed the determination of
three acidity constants. Moreover, the fluorescence intensity of the probe decreases as pH is
increased and is off above pH equal to 9. This effect is assigned to a PET process induced by
deprotonation of the amino group of the fluorescent platform. To improve the possibility of
application of this type of probe in cellular media it would be desirable to replace the imide
substituent with other groups that enhance water solubility but produce higher fluorescence
intensity at physiological pH conditions.
The fluorescence intensity of compound, N-3,4-HPO, is also quenched in the presence of
variable concentration of Fe3+, Cu2+ and Zn2+ and the effect is assigned to formation of the
corresponding metal ion complexes. As a drawback, the fluorescent probe was not found to be
selective to any of the three metal ions although it allows monitoring Cu2+ using EPR
spectroscopy at room temperature.
Acknowledgments
This work was supported by Fundação para a Ciência e a Tecnologia through projects
PTDC/QUI/67915/2006 and PEst-C/EQB/LA0006/2011. T. Moniz also thanks FCT the PhD
grant (SFRH/BD/79874/2011). We thank Dr. Andrea Carneiro from CeNTI, V.N. Famalicão,
for making available the CEM Discover microwave reactor. The Bruker Avance II 400
spectrometer is part of the National NMR network and was purchased under the framework of
the National Programme for Scientific Re-equipment, contract REDE/1517/RMN/2005, with
funds from POCI 2010 (FEDER) and (FCT).
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20

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Highlights
The conjugation of a naphthalimide with a pyridinone gave a new fluorescent
probe.
The conjugation reaction was successfully achieved by using microwave
irradiation.
Photophysical properties (absorption, fluorescence, quantum yield) were
determined.
Fe(III), Cu(II) and Zn(II) lead to a considerable fluorescence quenching effect.

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Fig 1. 4-amino-1,8-naphthalimide structure.

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Fig. 2 1,8-Naphthalimide modified 3,4-HPO (N-3,4-HPO).

ACCEPTED MANUSCRIPT
1.8
A
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1
3
N-3,4-HPO
300
400
500
600
λ
/ nm
500
400
300
200
100
0
B
N-3,4-HPO
3
1
350
400
450
500
550
/ nm
600
650
700
λ
Fig. 3. (A) UV-Vis spectra of 1,8-naphthalic anhydride (1) (black line), compound 3 (red line) and N-3,4-HPO (blue line) in DMSO
(T=25ºC) and concentration 7.5x10-5 M; (B) Fluorescence spectra of 1,8-naphthalic anhydride (1) (black line, λexc = 342 nm, λem
from 344-700 nm), compound 3 (red line, λexc = 348 nm, λem from 350-700 nm) and N-3,4-HPO (blue line, λexc = 437 nm, λem from
438-700 nm) in DMSO (T=25ºC, 600 V, 5 nm slits) and concentration 7.5x10-5 M.

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0.06
A
3
0.05
0.04
1
N-3,4-HPO
0.03
0.02
0.01
0.00
300
400
500
600
λ / nm
300
200
100
0
B
N-3,4-HPO
3
1
400
450
500
λ / nm
550
600
650
Fig. 4. (A) UV-Vis spectra of 1,8-naphthalic anhydride (1) (black line), compound 3 (red line) and N-3,4-HPO (blue line) in MOPS
(I=0.1 M NaCl, pH=7.4, T=25ºC) and concentration 3.0x10-6 M; (B) Fluorescence spectra of 1,8-naphthalic anhydride (1) (in black,
λexc = 342 nm, λem from 344-650 nm), compound 3 (in red, λexc = 348 nm, λem from 350-650 nm) and N-3,4-HPO (in blue, λexc = 439
nm, λem from 441-650 nm) in MOPS (pH=7.4, T=25ºC, 600 V, 5 nm slits) and concentration 3.0x10-6 M.

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0.7
pH = 1.60
0.6
0.5
0.4
0.3
0.2
0.1
0.0
pH = 2.77
pH = 3.34
pH = 4.26
pH = 5.39
pH = 6.30
pH = 7.33
pH = 8.37
pH = 9.22
pH = 10.13
250
300
350
400
/ nm
450
500
550
λ
Fig. 5. Absorption spectra of 4.0x10-5 M aqueous solution of N-3,4-HPO at variable pH and 25ºC.

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1
Fig. 6. Speciation diagram of N-3,4-HPO in aqueous solution.

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500
A
pH = 3.98
pH = 4.94
pH = 5.98
pH = 7.42
pH = 9.05
pH = 10.41
400
300
200
100
0
450
500
550
600
650
λ / nm
B
Fig. 7. (A) Fluorescence spectra of N-3,4-HPO at pH: 1.5, 4, 5, 6, 7.4, 9 and 10 (λexc=434 nm, λem from 441-650 nm; T= 25ºC, 590
V, 5 nm slits) and concentration 8.0x10-6 M; (B) Effect of pH on fluorescent properties of the ligand.