Highly selective 4-amino-1,8-naphthalimide based fluorescent photoinduced electron transfer (PET) chemosensors for Zn(ii) under physiological pH conditions

Article abstract of DOI:10.1039/b614529a

The design and synthesis of two novel fluorescent sensors based on the photoinduced electron transfer (PET) concept, 1 and 2, for the detection of zinc under competitive media is described. These sensors are based on the 4-amino-1,8-naphthalimide fluorophore, which has an absorption band centred at 450 nm and emits in the green with λ_max ～550 nm. By functionalizing the chromophore with a simple benzyl or ethyl-aryl based iminodiacetate receptor at the 4-position, both high selectivity and sensitivity were achieved for the sensing of Zn(ii) over other competitive transition and Group I and II metal ions. These sensors were also shown to be pH independent, with a pK_a of 2.3 being determined for 1, which allows these to be used in highly competitive pH media. Upon sensing of Zn(ii) the fluorescence emission spectrum is 'switched on' demonstrating the suppression of PET from the receptor to the fluorophore. For 1, the sensing of Zn(ii) was achieved with K_d = 4 nM when measured in pH 7.4 buffered solution, in the presence of 1.1 mM of EGTA. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b614529a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Highly selective 4-amino-1,8-naphthalimide based fluorescent photoinduced
electron transfer (PET) chemosensors for Zn(II) under physiological
pH conditions
Raman Parkesh,a,b T. Clive Leeb and Thorfinnur Gunnlaugsson*a
Received 5th October 2006, Accepted 27th October 2006
First published as an Advance Article on the web 16th November 2006
DOI: 10.1039/b614529a
The design and synthesis of two novel fluorescent sensors based on the photoinduced electron transfer
(PET) concept, 1 and 2, for the detection of zinc under competitive media is described. These sensors
are based on the 4-amino-1,8-naphthalimide fluorophore, which has an absorption band centred at
450 nm and emits in the green with kmax ∼550 nm. By functionalizing the chromophore with a simple
benzyl or ethyl-aryl based iminodiacetate receptor at the 4-position, both high selectivity and sensitivity
were achieved for the sensing of Zn(II) over other competitive transition and Group I and II metal ions.
These sensors were also shown to be pH independent, with a pKa of 2.3 being determined for 1, which
allows these to be used in highly competitive pH media. Upon sensing of Zn(II) the fluorescence
emission spectrum is ‘switched on’ demonstrating the suppression of PET from the receptor to the
fluorophore. For 1, the sensing of Zn(II) was achieved with Kd = 4 nM when measured in pH 7.4
buffered solution, in the presence of 1.1 mM of EGTA.
affecting neuron activity, it has also been observed that cortical
Introduction
neurons undergo cell death if exposed to intense Zn(II) levels and
The design and development of small molecules for sensing appli-
Zn(II) is now regarded as one of the most important co-factors
cations is of great current interest in supramolecular chemistry.1,2
In particular, luminescence sensing of ions and molecules has
become evermore important in medical diagnostics as such sensing
can give fast and reliable analysis of human wellbeing.3,4 Such
sensing has recently been demonstrated for analysis of blood or
serum samples for critical care analysis; where the concentration
of vital electrolytes can be determined instantly after sample
gathering.5 Using synthetically designed sensors, or chemosensors,
where a receptor moiety is conjugated directly or via a short
spacer into a luminescent moiety, such as a fluorescence tag or
metal based emitter, it is now possible to detect the presence
and concentration of most biologically relevant ions.6,7 In the
last few years, researchers have become particularly interested in
the physiological role of Zn(II).8,9 Zinc plays a vital role in both
physiological and metabolic pathways in the body. Biologically
relevant concentrations of Zn(II) in its mobile form, are thought
to be in the range of 1 fM in E. coli to almost 0.5 mM in mammalian
cells.10 It is anessential nutrient and stronglyinfluences cell division
and differentiation and hence is very important for the growth
and development of all forms of life.11 It plays a key role in the
synthesis of insulin and the pathological state of diabetes.12 It has
been reported that hyperglycaemia from either type I or type II
diabetes causes physiologically important losses of Zn(II) from the
body. It is known that Zn(II) serves as a mediator for cell–cell
signalling in the central nervous system (CNS), where the brain
tissue contains a high concentration of Zn(II).13 In addition to
in the regulation of apoptosis.14 Hence, the ability to selectively
detect Zn(II) at pH 7.4 in competitive media such as Ca(II), Mg(II)
and other transition metal ions, can allow a further understanding
of its physiological role in nature and disease.8,9,15,16
For some years we have been interested in the luminescence
sensing of both cations and anions using fluorescence and delayed
lanthanide luminescence sensing.17–19 Our interest in the sensing
of Zn(II) sprang from the fact that many of the examples in
the literature had significant drawbacks which included: being
synthetically challenging, possessing poor water solubility, pH
sensitivity within the physiological pH region, possessing low
quantum yield, or small luminescent enhancement between the
‘free’ and the ’complexed’ sensor, sensitive to group II ions and
as such, lacking in Zn(II) selectivity. With this in mind we set out
to design a new family of Zn(II) selective sensors which would
overcome all of these drawbacks.20
Our Zn(II) sensors discussed herein, are synthetically simple
and based on the photoinduced electron transfer (PET) principles
where a fluorophore is connected to a receptor by a short spacer.
Here, 4-amino-1,8,-naphthalimide was chosen as the fluorophore,
which has a strong absorption band in the visible region, emits
at long wavelengths with large Stokes shifts and possesses high
fluorescence quantum yield in aqueous media.21 Unlike the Zn(II)
sensors developed to date, many of which consist of the two
bis(2-pyridylmethyl)amine units as the Zn(II) receptor, our sensor
employs a phenyl iminodiacetate receptor.22 This ensures pH
independence in the physiological pH range at the same time as
providing high selectivity and excellent affinity for Zn(II) over
other biologically competitive metal ions. Employing the PET
principle is advantageous as, if correctly designed, the sensors
do not give rise to any fluorescence except upon coordination to
aSchool of Chemistry, Centre for Synthesis and Chemical Biology (CSCB),
Trinity College Dublin, Dublin 2, Ireland. E-mail: gunnlaut@tcd.ie;
Fax: +353 1 671 2826; Tel: +353 1 608 3459
bDepartment of Anatomy, Royal College of Surgeons in Ireland, St. Stephen’s
Green, Dublin 2, Ireland
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Zn(II), which ‘switches the emission on’.23 The PET Zn(II) sensors
presented herein, 1 and 2, are the results of our investigation.
such as refluxing 3 with 4-aminobenzylamine in DMF, dioxane
or ethanol all failed. Nevertheless, 4 was successfully synthesized
by heating 3 to near melting temperature (130 ◦C) followed by
subsequent addition of neat 4-aminobenzylamine. This resulted
in the formation of the desired product 4 within 30 minutes in
85% yield, after precipitation from water and a recrystallization
from ethanol. The synthesis of 5, the ethyl analogue of 4, was
accomplished using a similar protocol whereby 4-aminophenyl-
N-ethylamine was heated at 130 ◦C followed by precipitation and
recrystallization from 2-propanol, giving the desired product in
80% yield. Different methods were attempted for the alkylation
of 4 using ethyl bromoacetate, such as using K2HPO4 in refluxing
CH3CN, or DMF, but these resulted in the formation of mixture of
products that were difficult to separate. Similarly, using K2CO3 in
CH3CN gave a mixture of products. However, the desired product
was successfully formed by using K2CO3 in DMF and the target
molecule 6 was formed in high purity and in 90% yield after
recrystallization from 2-propanol. Similarly, 7 was formed using
the same N-alkylation method in a 90% yield. Finally the two PET
sensors 1 and 2 were formed, in a quantitative yield, by alkaline
hydrolysis of 6 and 7, respectively, using 3 M NaOH in CH3OH :
H2O (10 : 1, v/v). All the compounds were fully characterized
using conventional spectroscopic techniques. The aromatic region
of the 1H NMR (400 MHz, D2O) of 1 is shown in Fig. 1,
Results and discussion
Design and synthesis of 1–4
The design of 1 and 2 is based on the classical PET principle,
developed by de Silva.23,24 In our design we used a methylene
spacer for 1 and an ethylene spacer for 2. The rate of electron
transfer, from the electron rich receptor to the electron deficient
fluorophore is highly distance dependent. We would thus expect
1 to be a more efficient PET sensor than 2. Nevertheless, the
rationale behind 2 was that the ethylene spacer would impart
more photo-stability, as well as being more stable to metabolite
breakdown in vivo, which overall would lengthen its usability
for practical application.25 The synthesis of 1 and 2 is shown
in Scheme 1. For both sensors, the starting point was the N-
ethyl-4-amino-1,8-naphthalimide, 3, which was synthesized by
refluxing 6-bromo-1,8-naphthalic anhydride in 1,4-dioxane and
70% aqueous ethylamine (w/v), and obtained as an off-white solid
in 85% yield after precipitation from water. The next step was the
introduction of the spacer moieties in 1 and 2. The synthesis of the
benzyl derivative 4 was first attempted from 4-aminobenzylamine,
which would incorporate the spacer as well as the aniline part
of the receptor moiety in one step. However, the synthesis of
this molecule turned out to be problematic and several reactions
Fig. 1 Partial 1H NMR (400 MHz) of 2 in D2O. Protons a, b, c, e and g
are assigned to the naphthalimide fluorophore while d and f are those of
the aniline receptor.
Scheme 1 Synthesis of PET sensors 1 and 2.
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showing doublets centred at d 6.47 and d 7.22 ppm for the phenyl
protons and a doublet, a triplet and three doublets resonating at
7.81, 7.69, 5.58, 7.11 and 6.24 ppm for the naphthalimide protons.
A similar 1H NMR was observed for 2.
ICT band at the isosbestic point at 422 nm, the ICT emission was
observed at a long wavelength between 500–700 nm, with kem max
=
550 nm. The changes in the emission spectra of 1 are shown
in Fig. 3, and clearly demonstrate that the observed emission
is highly pH dependent; being ‘switched on’ upon acidification,
with large fluorescent enhancements. These changes were found
to be fully reversible, demonstrating the suppression of any PET
quenching from the electron rich receptor to the naphthalimide
fluorophore upon protonation of the aniline nitrogen, which
increases the oxidation potential of the receptors and hence makes
PET thermodynamically unfavorable.23,24 From the changes at
550 nm (insert in Fig. 3), a pKa of 3.20 0.1 was determined for 1.
This is in good agreement with the ground state pKa measurement,
and clearly demonstrates the pH independence of this sensor
within the physiological pH range. Hence, as the emission can
be considered to be ‘switched off’ at pH 7.5, any change in the
emission intensity as a function of added metal ions, e.g. Zn(II),
can be considered a direct measure of the binding of the ions to the
receptor at this pH. In a similar manner, the fluorescence emission
was monitored for 2 as a function of pH upon excitation at 453 nm.
As above, the emission was found to be highly pH dependent,
being ‘switched off ’ in alkaline solution and gradually ‘switched
on’ in acid solution below pH 5. From the changes at 554 nm
(kmax) a pKa = 2.10 0.1, was determined. This is considerably
less than that observed for 1. This may be due to the electronic or
conformational effects of the ethylene spacer. Nevertheless, these
results show that the emission from 2 is also pH independent in
the physiological pH range. These are highly desirable features for
any metal ion chemosensors for sensing in vivo.
Spectroscopic pH evaluation 1 and 2
The luminescent properties of both sensors were first investigated
as a function of pH in water in the presence of 135 mM KCl
(to maintain constant ionic strength). Both sensors were fully
water soluble. The absorption spectrum of 1, when recorded in
alkaline solution, showed two main spectral regions between 258–
293 nm, assigned to the p–p* transition, and a second region
centred at 440 nm (e = 18.92 × 103 M−1 cm−1) assigned to
the internal charge transfer (ICT) character of the fluorophore,
arising due to the push–pull nature of the donating amine and
the withdrawing diimide. Upon titration with acid, the 450 nm
band was hypsochromically shifted to shorter wavelengths, with
the formation of an isosbestic point at 422 nm, Fig. 2. This was
assigned to the protonation of the amino moiety of the receptor
and not that of the 4-amino moiety, which usually occurs at much
lower pH. For ideal PET sensors, such shifts do not usually occur,
due to the presence of the spacer between the fluorophore and
the receptor. However, the observed hypsochromic shift can be
explained by the ICT excited state of the naphthalimide, which
places a partial positive charge on the 4-amino moiety. This creates
a repulsive interaction with the protonated N-acetate moiety thus
shifting the absorption spectra to lower wavelengths.26 The plot of
absorbance at 450 nm against pH showed that only minor changes
occurred above pH 6.5, while the most significant changes were
observed between pH 3–5. From these latter changes a pKa of 3.2
0.1 was determined, using non-linear regression analysis. In the
same way the absorption spectra of 2 were monitored as function
of pH. In alkaline solution the main absorption band was observed
at 453 nm (e = 20.1 × 103 M−1 cm−1), with a lower intensity band
at 306 nm. Upon acidification, a ca. 20% decrease was observed
in the intensity of both bands, but no hypsochromic shift was
observed, possibly the presence of the longer ethylene spacer,
reduces the aforementioned repulsive interactions. Unfortunately,
these changes were too small for accurate pKa determination.
Fig. 3 Changes in the fluorescence emission spectra of 1 (1 lM) upon
changes in pH. Insert: The changes at 550 nm as a function of pH.
Spectroscopic evaluation 1 and 2 in the presence of various Group
II and transition metal ions
Having established the pH dependence of both sensors, we
evaluated the fluorescence and absorption dependence for both
1 and 2 (1 lM) towards Group II and transition metal ions such
as Zn(II), Cd(II), Hg(II), Cu(II), Co(II), Ni(II) and Fe(III), at pH 7.4
(20 mM HEPES) in the presence of 135 mM KCl. No fluorescence
emission changes were observed with Ca(II) or Mg(II), even at
10−2 M concentrations. Among the transition metal ions, Cu(II),
Fe(II) and Fe(III) did not modulate the fluorescence intensity,
and the emission remained ‘switched off ’. Minor fluorescence
enhancements were however, observed at higher concentrations
(>10−2 M) for Co(II), Ni(II), Hg(II) and Cd(II) (see later Fig. 5).
Fig. 2 Changes in the absorption spectra of 1 (1 lM) upon changes in
pH. Insert: The changes at alkaline and acidic pH.
The changes in the fluorescence emission spectra of both 1 and
2 were also monitored as a function of pH. Upon excitation of the
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However, in the presence of Zn(II) dramatic changes were observed
in fluorescence emission, which was ‘switched on’ as can be seen in
Fig. 4.
Zn(II). Using the value for e (determined above), the brightness
of the Zn(II) bound sensor can be determined (e × U) as 1.01 ×
106 Int M−1 cm−1.
Sensor 1 also functions as an ideal PET sensor as the ground
state was not modulated upon Zn(II) binding to the receptor, Fig. 5.
Some minor changes were observed at lower wavelengths that
can be assigned to the coordination of Zn(II) to the aniline unit.
However, these are too small for accurate binding constant deter-
mination. Similarly, no changes were observed in the absorption
spectra in the presence of other competitive metal ions.
The fluorescence response of 2 to Zn(II) was similar to that
observed for 1, Fig. 6. A broad emission band with a maximum at
554 nm was observed upon excitation at 453 nm. In the unbound
form, the sensor 2 was determined to have a quantum yield of
∼0.007 which increased to 0.14 in the presence of a saturating
amount of Zn(II) (5 lM), hence a fluorescence quantum yield
enhancement (Ubound/Ufree) of 20-fold was observed upon Zn(II)
binding. This is a good enhancement, given the longer spacer
length and shows that 2 can also function as a luminescent light-
switch for Zn(II). As in the case of 1, no shifts were observed in the
emission wavelength. From these changes a log b = 4.0 0.1 was
calculated for the 1 : 1 binding, which indicates that 2 has the same
affinity for Zn(II) as observed for 1. The extinction coefficient (e)
of the sensor was calculated to be 20.1 × 103 M−1 cm−1, with a
brightness of 4.02 × 105 Int M−1 cm−1 for the Zn(II) bound sensor.
These results clearly indicated that the two sensors fulfil the criteria
set out above for the development of highly selective Zn(II) sensing
in competitive media.
Fig. 4 Changes in the fluorescence emission spectra of 1 (1 lM)
upon addition of Zn(II) upon excitation at 422 nm, at pH 7.4. [Zn(II)]:
1.2 nM–1.5 mM. Insert: The changes at 550 nm as a function of
−log[Zn(II)], pZn.
Fig. 5 Absorption spectra response of 1 in the presence of increasing
concentration of Zn(II) (cf. Fig. 4) in HEPES buffer at pH 7.4.
No measurable changes were observed in the emission wave-
length, which indicates a PET mechanism. These changes rep-
resent the binding of the ion to the Zn(II) receptor, via the
carboxylates of the iminodiacetate and the nitrogen moiety, and
the concomitant suppression of any PET activities from the
receptor to the fluorophore. This is due to the increase in the
oxidation potential of the receptor upon Zn(II) binding, in a
similar manner to that observed for H+. From these measurements
the binding constant log b = 3.9 ( 0.1) was determine using the
least squares regression analysis program Sigma Plot. The changes
in the emission intensity suggest a simple equilibrium and 1 : 1
binding. The 1 : 1 binding was also determined using both Job
plot and Hill plot analysis. For the latter a Hill coefficient of 1.05
was calculated, which indicates the correct 1 : 1 stoichiometry.
For the free sensor 1 the fluorescence quantum yield (Ufree) under
these conditions was measured to be ca. 0.004, increased to 0.21
in the presence of 5 lM Zn(II), (Ubound). This is a fluorescence
enhancement of 56 fold upon Zn(II) binding, which shows that
the sensor can be considered as a luminescent light-switch for
Fig. 6 Changes in the fluorescence emission spectra of 2 (1 × 10−6 M)
upon addition of Zn(II) upon excitation at 455 nm, at pH 7.4. [Zn(II)] are
the same as in Fig. 4. Insert: The changes at 554 nm as a function of pZn
( = −log[Zn(II)]).
Selectivity of 1 and 2 for Zn(II) over other competitive ions
As briefly discussed above, the selectivity of both sensors towards
Zn(II) was found to be excellent. The fluorescence and absorption
responses of 1 and 2 to Ca2+ and Mg2+ were first investigated as
these are highly competitive ions in vivo. The addition of these
ions, even at high concentrations (>10−2 M), did not result in any
changes in the absorption and the fluorescence emission spectra.
Of the transition metal ions, only Cd(II) and Hg(II) gave rise
to any significant changes. Addition of Cd(II) to 1 resulted in
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no spectral shifts in the ground state spectrum. However, the
fluorescence titration of 1 with Cd(II) lead to a 10-fold increase in
fluorescence. The changes in emission were fitted to a 1 : 1 binding,
and a log b = 2.0 0.1 was determined from these changes. For
2, even higher concentrations of Cd(II) were required to achieve
any significant increase in the fluorescence emission, and a log
b = 1.8 0.1 was determined from these changes. Hence, it can be
concluded that both these sensors show minor changes with Cd(II).
Nevertheless, these concentrations are much higher than the level
of Cd(II) present in the body, and as such, will not interfere in
the measurement of the Zn(II) ions in important organs such as
the pancreas and brain. The responses towards to Hg(II) were
negligible and mirrored the Cd(II) response, with an overall 4-fold
enhancements being observed on titrations of 1 with Hg(II), while
Ni(II) gave even smaller enhancements. This clearly demonstrates
that the receptor has excellent affinity for Zn(II) over these ions.
To further evaluate the selectivity of 1 towards Zn(II) we carried
out competitive measurements. The results of these titrations are
shown in Fig. 7, where a 1 lM solution of 1 at pH 7.4 (20 mM
HEPES, 135 mM KCl) was measured after the addition of 5 lM
of each of the metal ions shown. These results clearly demonstrate
the selectivity for Zn(II) for 1 over the other metal ions, as on
all occasions emission intensity (recorded at 550 nm) increased
significantly upon addition of Zn(II). To the best of our knowledge,
1 is the first example of a Zn chemosensor to uphold such strict
Zn(II) selectivity.
Fig. 8 Changes in the fluorescence emission spectra of 1 upon addition of
Zn(II) upon excitation at 455 nm, at pH 7.4 (20 mM HEPES) and 135 mM
KCl and 1.1 mM EGTA.
As seen above, no significant changes were observed in the
absorption spectra. However, the fluorescence emission response
was significantly affected, being ‘switched on’ with fluorescence en-
hancements of almost 28-fold. From these changes a dissociation
equilibrium constant, Kd, was obtained by plotting the log[(IF
−
I
F min)/(IF max − IF)], where IF is the measured fluorescence after
each addition; IF min is the initial emission and IF max is the saturated
emission, versus the log of free Zn(II). A Hill plot was obtained,
with a log Kd = 8.3
0.1 and therefore a Kd = 4 nM, Fig. 9.
These results clearly demonstrate the affinity of 1 for free Zn(II)
concentrations and the versatility of our design.
Fig. 7 Relative fluorescence intensity responses of 1 to various metal
ions. Dark grey bars represent emission without Zn(II) for a particular
metal ion. Light grey bars represent the emissions upon addition of Zn(II)
to respective metal ion. The first bar shows the response to Zn(II) in the
absence of any competitive ions.
Fig. 9 Changes in the fluorescence emission of 1 (1 lM) at 550 nm as a
function of free Zn(II) at pH 7.4 (20 mM HEPES) and in the presence of
135 mM KCl and 1.1 mM EGTA. Insert: The fitting of the corresponding
Hill plot.
Conclusion
Spectroscopic evaluations of 1 and 2 towards free Zn(II) ions
The synthesis of two fluorescent water soluble PET sensors for
the sensing of Zn(II) has been achieved in a few steps and in high
yields. We have demonstrated that both sensors show ideal PET
behavior for Zn(II), where the absorption spectra do not change
to any significant degree upon binding to the ion. In contrast,
the emission spectra change dramatically; being enhanced, or
‘switched on’, significantly for both sensors. Importantly, both 1
and 2 show high selectivity for Zn(II) ions, even in the presence
of other competitive ions. All the ion titrations described herein,
were conducted at pH 7.4, where the emission for both sensors
was ‘switched off ’. Hence, these sensors are pH independent in the
physiological pH range. To the best of our knowledge these are the
Having established the high selectivity of both sensors towards
Zn(II), the ability of 1 to determine free Zn(II) concentration
was evaluated. Free Zn(II), or chelatable Zn(II), is found in high
concentrations in the brain, the nervous system and the pancreas.
To determine the affinity of 1 towards free Zn(II), a buffered
solution of 1 (1 lM) containing 135 mM KCl, 20 mM HEPES
and 1.1 mM ethylene glycol tetraacetic acid EGTA was prepared
and to this was added varying volumes of 1 mM ZnCl2 solution
giving free Zn(II) concentrations of 1, 3, 5, 9, 12, 20, 30, 45, 80, 90,
100, 120, 140, 160, 200, 220, 240 and 280 nM, and the absorption
and the fluorescence emission spectra recorded (Fig. 8).27
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first examples of such PET sensors for Zn(II) that give rise to high
selectivity for Zn(II) within the physiological pH range. We are
currently evaluating the biological application of these sensors in
greater detail, the outcome of this investigation will be published
in due course.†
under argon. After 10 minutes an excess of 4-aminobenzylamine
(3.01 g, 24.65 mmol) was added in a single portion. After 30 min
water (20 ml) was added to the reaction mixture, which resulted
in the formation of a yellow precipitate which was isolated by
filtration and recrystallized from ethanol to give 4 as light yellow
needles in 85% yield (1.48 g), mp = 198–200 ◦C. MS (ES+) m/z =
346 (M + H)+. Anal. calculated for C21H19N3O2: C, 73.03; H, 5.54;
N, 12.17. Found: C, 72.76; H, 5.63; N, 12.06. 1H-NMR (400 MHz,
DMSO-d6): d, 1.17 (t, 3H, J = 7.0 Hz, NCH2CH3), 4.04 (q, 2H,
J = 7.0 Hz, NCH2CH3), 4.54 (d, 2H, J = 5.2 Hz, CH2) 4.95 (brs,
2H, NH2), 6.54 (d, 2H, J = 8.0 Hz, Ar-H), 6.70 (d, 1H, J = 8.5 Hz,
Ar-H), 7.07 (d, 2H, J = 8.0 Hz, Ar-H), 7.68 (t, 1H, J = 7.5 Hz,
Ar-H), 8.18 (d, 1H, J = 8.5, Ar-H), 8.42 (brs, 1H, NH), 8.43 (d,
1H, J = 8.5 Hz, Ar-H), 8.74 (d, 1H, J = 8.5 Hz, Ar-H). 13C-NMR
(100 MHz, DMSO-d6): d, 163.53, 162.673, 150.54, 147.70, 133.92,
130.54, 129.37, 128.52, 127.93, 124.93, 124.27, 121.95, 120.26,
107.81, 104.51, 45.86, 34.21, 13.29. IR (mmax, KBr, cm−1): 3358,
2974, 2931, 2108, 1679, 1639, 1579, 1536, 1390, 1346, 1248, 1181,
1101, 1066, 910, 876, 773, 757, 694, 581.
Experimental
General
All solutions were prepared in deionized water. ZnCl2, CdCl2,
and HgCl2 (99%) were used for preparing stock solutions (1 M)
of Zn(II), Cd(II) and Hg(II). Zn(II), Cd(II) and Hg(II) solutions
of varying concentrations were prepared by the dilution of
appropriate amounts of 1 M stock solution with deionized water.
Absorption spectra were recorded on a Shimadzu diode array
spectrophotometer under the control of a Pentium IV-based
PC utilizing the manufacturer supplied software package. Flu-
orescence spectra were recorded on a Varian spectrofluorimeter
interfaced to the PC via an IEEE-488 (GPIB) card using Cary
Eclipse software. Excitation was provided by a 150 W Xe lamp
operating at a current of 5 A. Spectra were acquired in a 1 × 1 cm
quartz cuvette (3 mL) using a slit width of 5 nm and a scan rate
of 400 nm min−1. Both absorption and fluorescence spectra were
acquired at room temperature. Data were analyzed with either
Microsoft Excel or Sigma Plot. pH values of the solutions were
recorded with a calibrated glass electrode.
N-Ethyl-4-{4-[bis(ethoxycarbonylmethyl)amino]benzylamino}-
1,8-naphthalimide (6). To a solution of 4 (1.5 g, 4.34 mmol) in
dry DMF (50 mL) was added K2CO3 (1.67 g, 9.46 mmol) and
KI (1.57 g, 9.46 mmol). The resulting solution was stirred at room
temperature under argon. After 10 min ethyl bromoacetate (1.57 g,
9.46 mmol) was slowly added via a syringe, and the resulting
mixture was heated at 90 ◦C overnight. The solvent was then
evaporated under reduced pressure and the resulting residue was
taken into chloroform (30 mL) and washed with 1 M HCl (30 mL)
water (30 mL) and finally brine (30 mL). The organic layer was
then dried over Na2SO4, and evaporated to dryness under reduced
pressure. The resulting solid was recrystallized from ethanol to
give 6 in 90% yield (2.02 g) as a light yellow solid, mp = 180–
Synthesis
N-Ethyl-4-bromo-1,8-naphthalimide (3). 4-Bromo-1,8-naph-
thalic anhydride (1.5 g, 5.42 mmol) was dissolved in 1,4-dioxane
(50 mL) and ethylamine (0.35 g, 6.49 mmol) was added. The
resulting solution was refluxed for 2 hours after which the solution
was poured into ice–water. The product 3 was collected by suction
filtration and washed with water and dried over P2O5. This gave
3 in 85% (1.66 g), mp = 162–164 ◦C (lit. 160–161 ◦C). 1H-NMR
(400 MHz, CDCl3): d, 1.35 (t, 3H, J = 7.0 Hz, NCH2CH3), 4.23–
4.29 (q, 2H, J = 7.0 Hz, NCH2CH3), 7.85 (t, 1H, J = 7.5 Hz,
Ar-H), 8.05 (d, 1H, J = 7.5 Hz, Ar-H), 8.42 (d, 1H, J = 7.5 Hz,
Ar-H), 8.58 (d, 1H, J = 7.5 Hz, Ar-H), 8.61 (d, 1H, J = 7.5 Hz,
Ar-H). 13C-NMR (100 MHz, CDCl3): d, 162.97, 132.77, 131.51,
130.72, 130.62, 130.15, 129.75, 128.52, 127.61, 122.70, 121.85,
35.20, 12.80.
◦
182 C. MS (ES+) m/z = 518 (M + H)+. Anal. calculated for
C29H31N3O6: C, 67.30; H, 6.04; N, 8.12. Found: C, 67.03; H,
1
6.02; N, 7.91. H-NMR (400 MHz, DMSO-d6): d, 1.16 (m, 9H,
NCH2CH3, NCH2CO2CH2CH3), 4.01–4.11 (m, 6H, NCH2CH3,
NCH2CO2CH2CH3), 4.15 (s, 4H, NCH2CO2CH2CH3), 6.5 (d, 2H,
J = 8.0 Hz, Ar-H), 6.67 (d, 1H, J = 8.5 Hz, Ar-H), 7.21 (d,
2H, J = 8.0 Hz, Ar-H), 7.69 (t, 1H, J = 8.0 Hz, Ar-H), 8.17
(d, 1H, J = 8.0 Hz, Ar-H), 8.37 (brs, 1H, NH), 8.44 (d, 1H,
J = 7.0 Hz, Ar-H), 8.73 (d, 1H J = 8.0 Hz, Ar-H). 13C-NMR
(100 MHz, DMSO-d6): d, 170.54, 163.55, 162.69, 150.44, 146.84,
133.98, 130.65, 129.32, 128.52, 127.94, 126.53, 124.42, 121.89,
120.89, 111.86, 107.84, 104.56, 60.40, 52.66, 45.35, 34.26, 14.08,
13.30. IR (mmax, KBr, cm−1): 3386, 2974, 2929, 1747, 1727, 1685,
1616, 1590, 1573, 1541, 1523, 1450, 1397, 1367, 1251, 1183, 1102,
1027, 819, 801, 771, 760, 605, 585.
N-Ethyl-4-(4-aminobenzylamino)-1,8-naphthalimide (4). Com-
pound 3 (1.5 g, 4.93 mmol) was heated with stirring at 130 ◦C
† To evaluate the ability of these sensors to detect Zn(II) in biological
samples, preliminary histological staining of human pancreas tissue using
1 has been carried out. A standard staining protocol was followed, after
which glass slides containing pancreas tissue sections were immersed for
10 minutes in a bath containing 5 lM buffered solution of 1 at pH 7.4. The
glass slide viewed under a green 546 nm epifluorescence microscope and
the image collected. The control tissue section, without staining with 1,
was also recorded. In both cases, the dull green colour observed is due to
autofluorescence from proteins and light scattering. However, the sample
stained with 1, showed areas within the tissue that were highly luminescent
clearly demonstrating the ability of 1 to detect Zn(II). These preliminarily
studies clearly show promising results as 1 labels the area within the tissue
which is most likely to contain granular chelatable zinc. We are currently
evaluating the ability of these sensors to monitor Zn(II) in other biological
media such as cells.
Disodium N-Ethyl-4-{4-[bis(carboxylatomethyl)amino]benzyl-
amino}-1,8-naphthalimide (1). To a solution of 6 (1.43 g,
2.76 mmol) in dry ethanol (50 mL) was added NaOH (0.300 g,
7.5 mmol) in 1 mL of water. The resulting mixture was refluxed for
2 hours. Upon cooling to room temperature the desired product
was obtained as a yellow solid in 80% (1.33 g), mp = 310 ◦C
(decomp.). MS (ES+) m/z = 506 (M + H)+. Anal. calcd for
C25H21N3Na2O6·2H2O: C, 55.46, H, 4.65; N, 7.76. Found: C, 55.29;
1
H, 4.23; N, 7.72. H-NMR (400 MHz, D2O): d, 1.09 (t, 3H, J =
7.0 Hz), 3.75–3.79 (m, 6H), 4.28 (d, 2H), 6.24 (d, 2H, J = 8.8 Hz),
6.47 (d, 2H, J = 8.8 Hz), 7.11 (t, 1H, J = 7.6 Hz), 7.22 (d, 2H,
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J = 8.2 Hz), 7.58 (d, 1H, J = 8.8 Hz), 7.69 (d, 1H, J = 8.2 Hz),
7.81 (d, 1H, J = 7.6 Hz). 13C-NMR (400 MHz, D2O): d, 179.06,
164.31, 163.28, 158.55, 149.69, 147.69, 133.50, 129.84, 128.70,
124.20, 122.93, 118.79, 117.80, 111.13, 105.52, 55.21, 45.69, 34.94,
11.99. IR (mmax, KBr, cm−1): 3525, 3382, 2875, 1685, 1634, 1547,
1560, 1435, 1394, 1368, 1350, 1314, 1249, 1179, 1130, 1106, 1063,
979, 911, 877, 821, 769, 756, 705, 587.
2.76 mmol) in dry ethanol (50 mL) was added NaOH (0.30 g,
7.5 mmol) in 1 mL of water. The resulting solution was refluxed for
2 hours. Upon cooling, the desired product was obtained as a yel-
low solid in 95% yield (1.33 g), mp = 320 ◦C (decomp.). MS (ES+)
m/z = 520 (M + H)+. Anal. calculated for C26H23N3Na2O6·2H2O:
C, 55.46, H, 4.65; N, 7.76. Found: C, 55.29; H, 4.23; N, 7.72. 1H-
NMR (400 MHz, D2O): d, 1.09 (t, 3H, J = 7.3 Hz, NCH2CH3),
2.76–2.79 (m, 2H, CH2), 3.29–3.33 (m, 2H, CH2), 3.71–3.79 (m,
6H, NCH2CH3, NCH2CO2Na), 6.16 (d, 2H, J = 8.8 Hz, Ar-H),
6.44 (d, 2H, J = 8.0 Hz, Ar-H), 6.98 (t, 1H, J = 8.1 Hz, Ar-H), 7.08
(d, 2H, J = 8.0 Hz, Ar-H), 7.47–7.53 (m, 2H, Ar-H), 7.70 (d, 1H,
J = 8.2 Hz, Ar–H). 13C-NMR (100 MHz, D2O): d, 179.24, 164.23,
163.20, 149.68, 149.87, 133.49, 129.85, 129.07, 126.98, 125.94,
122.83, 118.78, 117.68, 111.29, 105.22, 102.94, 55.24, 43.79, 34.86,
32.68, 32.37, 11.96. IR (mmax, KBr, cm−1): 3374, 2933, 1906, 1854,
1680, 1638, 1546, 1519.51, 1524, 1435, 1399, 1351, 1311, 1253,
1188, 1110, 1066, 978, 919, 879, 811 772, 759, 700, 580.
N-Ethyl-4-[2-(4-aminophenyl)ethylamino]-1,8-naphthalimide (5).
Compound 3 was heated with stirring at 130 ◦C under argon
for 10 minutes, after which 4-ethylaminobenzylamine (3.01 g,
24.65 mmol) was added via a syringe. The reaction mixture was
kept stirring for 30 minutes, followed by the addition of water
(20 mL which resulted in the formation of a yellow precipitate
which was isolated by filtration and recrystallized from 2-propanol
to give 4 as light yellow needles in 85% yield (1.45 g), mp = 200–
◦
202 C. MS (ES+) m/z = 360 (M + H)+. Anal. calculated for
C22H21N3O2: C, 73.52; H, 5.89; N, 11.69; Found: C, 72.76; H, 5.63;
1
N, 12.06. H-NMR (400 MHz, DMSO-d6): d, 1.17 (t, 3H, J =
Acknowledgements
7.0 Hz, NCH2CH3), 2.81–2.84 (t, 2H, J = 8.2 Hz, CH2), 3.45–
3.54 (m, 2H, CH2), 4.04 (q, 2H, J = 7.0 Hz, NCH2CH3), 4.89 (d,
2H, J = 5.2 Hz), 6.51 (d, 2H, J = 8.2 Hz, Ar-H), 6.83 (d, 1H, J =
8.8 Hz, Ar-H), 7.13 (d, 2H, J = 8.2 Hz, Ar-H), 7.68 (t, 1H, J =
8.2 Hz, Ar-H), 7.84 (brs, NH), 8.28 (d, 1H, J = 8.2, Ar-H), 8.44
(d, 1H, J = 7.5 Hz, Ar-H), 8.66 (d, 1H, J = 8.2 Hz, Ar-H). 13C-
NMR (100 MHz, DMSO-d6): d, 163.53, 162.69, 150.42, 146.94,
134.24, 130.58, 129.38, 129.16, 125.94, 124.22, 121.87, 121.88,
We thank TCD, RCSI and HRB for financial support.
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a solution of 5 (1.5 g,
4.2 mmol) in dry DMF (50 ml) was added K2CO3 (1.30 g,
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◦
195–197 C. MS (ES+) m/z: 532 (MH)+. Anal. calculated for
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CH2), 3.52–3.54 (m, 2H, CH2), 4.04–4.13 (m, 6H, NCH2CH3,
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(d, 2H, J = 8.5 Hz, Ar-H), 7.67 (t, 1H, J = 8.0 Hz), 7.85 (brs,
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170.61, 163.54, 162.71, 150.41, 146.26, 134.25, 130.60, 129.40,
129.34, 128.52, 127.58, 124.26, 121.89, 120.13, 111.90, 107.69,
103.94, 60.35, 52.75, 44.84, 34.25, 32.89, 14.12, 13.32. IR (mmax
,
KBr, cm−1): 3378, 2974, 2929, 1753, 1731, 1686, 1581, 1543, 1524,
1430, 1395, 1367, 1296, 1181, 1103, 1062, 972, 911, 808, 770, 758,
655, 587.
N -Ethyl-4-(2-{4-[bis(carboxylatomethyl)amino]phenyl}ethyl-
amino)-1,8-naphthalimide (2). To a solution of 7 (1.43 g,
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