A new use for an old molecule: N-phenyl-2-(2-hydroxynaphthalen-1- ylmethylene)hydrazinecarbothioamide as a ratiometric 'Off-On' fluorescent probe for iron

Article abstract of DOI:10.1016/j.tetlet.2011.11.126

N-Phenyl-2-(2-hydroxynaphthalen-1-ylmethylene)hydrazinecarbothioamide has been investigated as a fluorescent sensor for the determination of Fe(III) in aqueous solutions. The probe was prepared by the facile Schiff base condensation of 2-hydroxy-1-napthal

Full text of DOI:10.1016/j.tetlet.2011.11.126

Tetrahedron Letters 53 (2012) 670–673
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A new use for an old molecule:
N-phenyl-2-(2-hydroxynaphthalen-1-ylmethylene)hydrazinecarbothioamide
as a ratiometric ‘Off–On’ fluorescent probe for iron
b
a
a
a
María José Casanueva Marenco , Colin Fowley , Barry W. Hyland , Graham R.C. Hamilton ,
b
a,
⇑
Dolores Galindo-Riaño , John F. Callan
Department of Pharmacy and Pharmaceutical Sciences, School of Biomedical Sciences, The University of Ulster, Northern Ireland BT52 1SA, United Kingdom
Department of Analytical Chemistry, Faculty of Sciences, University of Cadiz, Campus Rıo S. Pedro, 11510 Puerto Real, Cadiz, Spain
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Phenyl-2-(2-hydroxynaphthalen-1-ylmethylene)hydrazinecarbothioamide has been investigated as a
fluorescent sensor for the determination of Fe(III) in aqueous solutions. The probe was prepared by the
facile Schiff base condensation of 2-hydroxy-1-napthaldehyde with N-phenylhydrazinecarbothioamide.
The sensor displayed good selectivity for Fe(III) when tested against a range of biologically and environ-
mentally important cations. A concentration dependent increase in the emission of two fluorescent bands
at 425 and 495 nm was observed upon increasing Fe(III) addition resulting in a linear ratiometric
Received 27 October 2011
Revised 15 November 2011
Accepted 25 November 2011
Available online 2 December 2011
Keywords:
Fluorescence
Sensor
response in the 17–37
lM range. The binding stoichiometry was confirmed as 1:1 (host/guest) with
the binding constant (logb) calculated as 4.56.
Ó 2011 Elsevier Ltd. All rights reserved.
Iron(III)
Ratiometric
Iron is an essential element for normal physiological function-
ing as it plays an important role in many cellular processes includ-
ing energy generation, oxygen transport and DNA synthesis. The
prone, deferasirox and deferoxamine are approved for use in iron
chelation therapy. However, in order to administer the optimum
6
1
dose of these agents it would first be advantageous to determine
the level of excess iron present. As a result, there is a need for
the development of rapid detection systems that can determine
the concentration of iron in aqueous based solutions. In this capac-
ity, fluorescence based methods for analyte detection remain pop-
ular due to their high sensitivity, rapid response rate and relative
ability of iron to redox cycle between two stable forms, Fe(II) and
Fe(III), enables it to function as both an electron donor and accep-
tor, a property that makes it suitable as a co-factor in many differ-
ent enzymes critical for life.2 However, iron overload can cause
deleterious conditions such as b-thalassaemia and Friedreich’s
ataxia where the presence of excess iron leads to the generation
7
inexpense.
3
of reactive oxygen species (ROS) via the Fenton reaction. ROS
Thiosemicarbazones have been studied extensively as iron che-
lators in the treatment of cancer. Their high affinity for iron makes
them ideal for sequestering iron at the active site of the RR enzyme
can induce cell death by interacting with many important biomol-
ecules through a series of chemical reactions resulting in DNA oxi-
4
8
dation, mitochondrial damage and lipid peroxidation. Iron is also
thus inhibiting its function. A large family of thiosemicarbazone
essential for the catalytic activity of ribonucleotide reductase (RR),
the enzyme used in the rate determining step of DNA synthesis. As
cancer cells proliferate at a faster rate than normal cells, these cells
derivatives have been developed and investigated for their chemo-
therapeutic potential including those derived from 2-hydroxy-1-
naphthaldehyde. Given the high affinity these molecules have
for Fe(III) and that they possess an inherent fluorophore within
their structure, it seems sensible to investigate these compounds
as potential fluorescent probes for iron. Surprisingly, to date, the
ability of these agents to also function as self indicating fluorescent
sensors for iron has been overlooked.
9
5
tend to have a greater requirement for iron than healthy cells.
Thus iron sequestration has also been a target in certain cancer
3
chemotherapies.
To counter the problems of iron overload, iron chelation therapy
utilises ligating drugs that strongly bind to intra- and extracellular
iron promoting the excretion and subsequent depletion of toxic
In this Letter, we synthesise the simplest member of this family,
N-phenyl-2-(2-hydroxynaphthalen-1-ylmethylene)hydrazinecarbo-
3
iron concentrations in the body. Currently, three drugs, deferi-
9
thioamide (which in this Letter is designated NT) and investigate
its ability to function as a fluorescent probe for Fe(III) in aqueous
based solution.
⇑
Corresponding author. Tel.: +44 2870123059; fax: +44 2870123509.
E-mail address: j.callan@ulster.ac.uk (J.F. Callan).
0
040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.11.126

M. J. C. Marenco et al. / Tetrahedron Letters 53 (2012) 670–673
671
S
O
N
OH
S
N
H
OH
N
H
+
NH2
N
H
N
H
Scheme 1. Synthesis of NT from 2-hydroxy-1-naphthaldehyde and 4-phenylthio-semicarbazide.
The target compound NT was synthesised in one step by the
facile Schiff base condensation reaction of 2-hydroxy-1-napthalde-
hyde with N-phenylhydrazinecarbothioamide (4-phenylthiosemic-
arbazide) (Scheme 1) and was produced in a 65% yield after
1
0
recrystallisation from ethanol. The UV–Vis spectrum of NT, re-
corded in a THF/H O (9:1) HEPES buffered solvent system (pH
.4) is shown in Figure 1 and is characterised by a broad absorption
2
7
from 210 to 450 nm. Within this broad absorption profile there is
evidence of four main peaks with kmax values of 210, 245, 320
⁄
⁄
and 370 nm. The 210 and 245 nm peaks are consistent with
transitions of the aromatic rings, the 320 nm peak with a
p
p
–
–
p
p
transition of the C@N group while the 370 nm band reflects an
intramolecular charge transfer (CT) band of the entire conjugated
1
1
molecule. When excited at 370 nm in the same solvent system
the fluorescence spectrum exhibited two main bands in the visible
region with kmax values of 425 and 485 nm (Fig. 2a). The presence
of two bands in the fluorescence spectrum enables the possibility
Figure 1. Absorbance spectrum of NT recorded in a THF/H
buffered at pH 7.4. [NT] = 10 M.
2
O (9:1) solvent system
l
2
Figure 2. (a) Fluorescence spectra of NT (10 lM) in the absence and presence of different metal salts (25 lM) recorded in a THF/H O (9:1) solvent system buffered at pH 7.4.
Excitation wavelength = 375 nm (b) selectivity plot for NT where IÀIo/Io is plotted against each metal ion (I is the intensity in the presence of metal ion and Io the intensity in
the absence of metal ion).

6
72
M. J. C. Marenco et al. / Tetrahedron Letters 53 (2012) 670–673
(
a)
[
Fe (III)] = 37 µM
NH
HN
N
S
[
Fe(III)] = 0 µM
Fe
O
H
Figure 3. Proposed binding mechanism between NT and Fe3+
.
(
b)
of ratiometric analysis by comparing the ratio of the intensities of
the two bands as a function of analyte concentration. This is pre-
ferred over single wavelength analysis as the method is free from
the errors associated with receptor concentration, photobleaching
12
and environmental effects.
The selectivity of NT as a fluorescent probe for Fe3+ was tested
in a THF/H O (9:1) solvent system buffered at pH 7.4 by incubating
NT (10 M) with a range of environmentally and biologically
important metal ions (25 M) as their chloride salts. The addition
2
l
l
+
+
2+
+
2+
2+
2+
2+
2+
2+
+
of Li , Na , Mg , K , Ca , Mn , Co , Ni , Zn , Sr , Ag and
Ba caused virtually no change to the fluorescence spectrum of
2
+
2
+
NT. The addition of Pb caused a minor increase in both bands
2
+
while the addition of Cu led to a slight increase of the 425 nm
band with a significant quench of the 485 nm band (Fig. 2a). How-
Figure 4. (a) The concentration dependent increase in fluorescence of NT upon
3+
increasing [Fe ]. (b) Plot of the ratiometric fluorescence intensity of NT (I485/I425
)
against Fe3 concentration. [NT] = 10
+
lM, solvent: THF/H O (9:1) buffered at pH 7.4.
3
+
2
ever, the addition of Fe resulted in a significant increase of both
bands with a 104% increase for the 425 nm band and a 194% in-
crease for the 485 nm band. When a plot of the intensity at
4
85 nm was performed for each metal ion, excellent selectivity
Finally, the effect of solution pH on the fluorescence intensity of
NT was investigated by recording the fluorescence spectra over a
range of different pH values. The emission intensity at 485 nm
was plotted as a function of pH and this plot is shown in Figure 6.
These results illustrate that the fluorescence intensity remains rel-
atively constant from pH 2–9, but increases dramatically from pH
10–12. This large increase in fluorescence is most likely caused by
the deprotonation of the naphthalene hydroxy group of NT that
otherwise leads to a non-radiative decay from the excited state
3
+
was observed for Fe over all the other tested ions (Fig. 2b).
The reason for the large fluorescent enhancement upon the
addition of iron can be explained as follows: iron is known to form
a tridentate chelate with thiosemicarbazones such as NT (Fig. 3),
where the oxygen lone pair from the naphthyl hydroxy, the imine
nitrogen lone pair and the thiosemicarbazone sulfur lone pair, all
bind strongly to the Fe ion. This binding event prevents the ra-
pid isomerisation of the C@N group that otherwise happens in the
absence of Fe(III) and which leads to non-radiative decay of the ex-
cited state. Examples of ‘Off–On’ fluorescence sensors for Fe(III)
are rare given in the redox nature of this ion as it typically
quenches the excited state of organic fluorophores by electron
transfer processes when brought into close proximity of the fluoro-
phore. In the case of NT, the enhancement in fluorescence that
arises as a result of the inhibition of the C@N isomerisation process
is greater than the quenching ability of Fe3 and an ‘Off–On’ re-
sponse was observed.
3
+
13
1
1
by vibrationally coupling it to water. As this hydroxy group is
1
4
3+
also involved in the binding of the Fe ion by NT, it is also probable
that this interaction may also contribute to the fluorescence
enhancement observed upon the interaction of NT and Fe3+, as this
binding event would reduce the ability of the hydroxy group vibra-
1
5
a
tionally coupling to water. The pK of this group was calculated to
+
To determine the sensitivity range over which NT can measure
Fe(III), a series of solutions were prepared in which the concentra-
3
+
tion of NT was kept constant but the concentration of Fe was
gradually increased. The fluorescence of each solution was mea-
sured and the ratiometric intensity (I485/I425) plotted against
concentration (Fig. 4). Excellent linearity was observed in the
1
7–37
lM range indicating that NT can function as a ratiometric
‘
Off–On’ fluorescent sensor in this range. To confirm the binding
3+
16
stoichiometry between NT and Fe a Job plot was performed.
The results of this plot are shown in Figure 5 and indicate the
formation of a 1:1 (host/guest) complex. Based on a 1:1 binding
stoichiometry the binding constant (logb) was calculated to be
4
(
.56 using the equation log(FmaxÀF)/(F/Fmin) = log[cation]Àlogb,
where Fmax is the maximum fluorescence intensity, Fmin the min-
imum fluorescence intensity and F the measured fluorescence
3
+
3
+
F
i
g
u
r
e
5
.
(
a
)
J
o
b
p
l
o
t
f
o
r
N
T
a
n
d
F
e
i
l
l
u
s
t
r
a
t
i
n
g
t
h
e
f
o
r
m
a
t
i
o
n
o
f
a
1
:
1
(
N
T
/
F
e
)
1
7
intensity).
complex.

M. J. C. Marenco et al. / Tetrahedron Letters 53 (2012) 670–673
673
References and notes
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0. UV and Fluorescence Spectroscopy: Absorbance measurements were recorded on
a Varian Cary 50 Spectrometer using 10 mm quartz cuvettes. Fluorescence
measurements were recorded on a Cary Eclipse fluorimeter using 10 mm
quartz cuvettes. Excitation slit size was 5 nm and emission slit size was 5 nm.
Synthesis of NT: 2-hydroxy-1-naphthaldehyde (0.5 g, 2.9 mmol) was added to
N-phenylhydrazinecarbothioamide (0.49 g, 2.9 mmol) in DMF (15 mL) at room
temperature. After stirring for 18 h, the solvent was evaporated under reduced
pressure and the crude product recrystallised from hot EtOH. The resulting
Figure 6. Plot of fluorescence against pH for NT. [NT] = 10 lM.
be 10.62 using a derivation of the Henderson Hasselbach equation
by plotting Àlog(FmaxÀF)/(FÀFmin) against pH.18 Importantly, how-
ever, the fluorescence–pH titration showed that the emission
intensity of NT remained constant in the physiological range.
1
yellow crystals were dried in vacuo to yield 0.61 g of 1 (65% yield).
(500 MHz, DMSO-d ): 6.95 (2H, m, Ar-H), 7.14 (3H, m, Ar-H), 7.32 (3H, m, Ar-
H), 7.61 (2H, m, Ar-H), 8.23 (1H, s, Ar-H), 8.90 (1H, s, CH@N), 9.80 (1H, s, NH),
H NMR
In summary, we have utilised NT as an ‘Off–On’ ratiometric
6
fluorescence sensor for Fe3+ operable in semi aqueous solution.
1
1
0.34 (1H, s, OH), 11.50 (1H, s, NH). 13C NMR (125 MHz DMSO-d
19.0, 124.0, 125.6, 128.6, 129.2, 132.1, 133.1, 139.7, 144.0, 157.1. MS:
SO = 321.4, found 321.1.
6
): 110.2,
This compound displayed good selectivity for Fe3 when tested
+
against a range of physiological and environmentally important
15 3
Calculated for C18H N
1
1
1. Issa, R. M.; Khedr, A. M.; Rizk, H. J. Chin. Chem. Soc. 2008, 55, 875–884.
2. Singh, N.; Kaur, N.; Mulrooney, R. C.; Callan, J. F. Tetrahedron Lett. 2008, 49,
ions and was sensitive in the 17–37 lM range. The fluorescence
enhancement upon binding Fe3 was attributed to a cancellation
of the C@N isomerisation that otherwise leads to non-radiative de-
cay of the excited state. The binding stoichiometry was confirmed
+
6
690–6692.
13. Antholine, W.; Knight, J.; Whelan, H.; Petering, D. Mol. Pharmacol. 1977, 13, 89–
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14. Bhardwaj, V. K.; Pannu, A. P. S.; Singh, N.; Hundal, M. S.; Hundal, G. Tetrahedron
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15. (a) Fabbrizzi, L.; Poggi, A. Chem. Soc. Rev. 1995, 24, 197–202; (b) Singh, N.;
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N.; Kaur, N.; Dunn, J.; MacKay, M.; Callan, J. F. Tetrahedron Lett. 2009, 50, 953–
9
3
+
as 1:1 (NT/Fe ) and the binding constant logb was determined as
.56. Examples of enhancement based ratiometric fluorescence
2
4
3
+
sensors for Fe are extremely rare and to the best of our knowl-
edge, NT is the first reported example of an enhancement based
fluorescence probe for Fe3 that operates via the suppression of
C@N isomerisation. In addition, we believe that this provides a
good example of how already synthesised molecules can be uti-
lised for a different purpose. We are currently investigating other
known iron-chelating compounds for their potential as optical iron
sensors.
956.
+
16. Espada-Bellido, E.; Dolores Galindo-Riano, M.; Garcia-Vargas, M.;
Narayanaswamy, R. Appl. Spectrosc. 2010, 64, 727–732.
1
1
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