A Zn(II) ion selective fluorescence sensor that is not affected by Cd(II)

Article abstract of DOI:10.1016/j.tetlet.2007.08.040

Comparison of sensors sodium-2,6-diamino-(N,N,N′,N′-tetraacetate)-4-methylanisole 1 and sodium-2,6-diamino-(N,N,N′-triacetate)-4-methylanisole 2 reveal that the loss of an acetyl group in 2 leads to a more selective Zn(II) induced fluorescence enhancement

Full text of DOI:10.1016/j.tetlet.2007.08.040

Tetrahedron Letters 48 (2007) 7316–7319
A Zn(II) ion selective fluorescence sensor that is not
affected by Cd(II)
Julius N. Ngwendson and Anamitro Banerjee*
Department of Chemistry, University of North Dakota, Grand Forks, ND 58202-9024, USA
Received 27 July 2007; revised 8 August 2007; accepted 9 August 2007
Available online 15 August 2007
Abstract—Comparison of sensors sodium-2,6-diamino-(N,N,N0,N0-tetraacetate)-4-methylanisole 1 and sodium-2,6-diamino-
(N,N,N0-triacetate)-4-methylanisole 2 reveal that the loss of an acetyl group in 2 leads to a more selective Zn(II) induced fluorescence
enhancement and shows no response to any other metal ions including Cd(II). Structural modifications and AM1 calculations indi-
cate that the sensor uses the three acetyl groups and the 3° amino nitrogen for binding the metal ion. AM1 calculations imply a
trigonal bipyramidal coordination for Zn(II) with the solvent molecule occupying one of the axial positions.
Ó 2007 Elsevier Ltd. All rights reserved.
Over 300 enzymes and proteins require zinc to carry out
their biological functions.1 For example, the zinc sites in
protein domains play structural catalytic, and cocata-
lytic roles.2 Zinc is essential for the regulation of DNA
synthesis during the proliferation and differentiation of
cells.3 Zinc is also suspected to play a role in neurologi-
cal disorders such as Parkinson’s disease, Alzheimer’s
disease, amyotrophic lateral sclerosis, and epileptic
seizures.4 Environmental impact of Zn ion in sea water
due to human activity is being studied.5 Zinc ion selec-
tive fluorescence sensors play an important role in
studying these processes.
O
O
O
O
H
H
H
R N
2
NR
N
NR
N
N
NR
2
2
2
R
R
R
4
1
2
3
R = CH CO Na
2
2
Chart 1. Structure of compounds 1–4.
In this Letter, we present compound 2 as a Zn(II) ion
selective sensor that itself has minimal fluorescence,
but shows approximately twenty-fivefold enhancement
of the fluorescence intensity in the presence of Zn(II),
while showing no response to any other metal ions
including Cd(II).
Several fluorescence sensors for Zn(II) ion have been
reported in the literature.5a,6 Unfortunately, sensors that
can selectively detect Zn(II) ion without showing any
response to Cd(II) are extremely rare. Selective detection
of Zn(II) in the presence of Cd(II) becomes important in
environmental matrix when both the ions are likely to be
present.5 Recently, we reported sodium-2,6-diamino-
(N,N,N0,N0-tetraacetate)-4-methylanisole 1 (Chart 1) as
a Zn(II) selective fluorescence sensor that can be synthe-
sized in three simple steps.7 When the sensor is excited at
300 nm, it has an emission maximum at 386 nm. In the
presence of Zn(II) ion, the sensor shows a tenfold
enhancement in emission intensity. This sensor also
undergoes approximately fivefold increase in the fluores-
cence intensity in the presence of Cd(II).
Semi-empirical AM1 calculations using SPARTAN 6 of 1–
Zn(II) complex indicates that only three acetate groups
of the sensor are involved in binding the metal ion
(Fig. 1). This was a surprising result given the geometric
Zn+2
+2
-O2C
-O2C
CO2
-
Zn
O
-
N
N
CO2
1-Zn(II)
Keywords: Zinc sensors; Fluorescence sensors.
*
Figure 1. Energy minimized structure of 1–Zn(II) complex as predicted
Corresponding author. Tel.: +1 701 777 3941; fax: +1 701 777
by AM1 calculations.
2331; e-mail: abanerjee@chem.und.edu
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.08.040

J. N. Ngwendson, A. Banerjee / Tetrahedron Letters 48 (2007) 7316–7319
7317
constraints of involving two groups in meta positions in
1.2
1
binding the metal ion. The presence of the methoxy
group between them also implies that the metal ion must
be held above the aromatic ring. Also, only three of the
acetyl groups interact with the metal, which lies more to
the side that contributes two of the acetyl groups. It ap-
pears from the calculations that the Zn(II) ion is located
roughly above the 3° amino nitrogen and is coplanar
with the three carboxylate oxygen atoms. When a mole-
cule of water was added, it occupied a position directly
above the metal ion without affecting the positions of
other atoms. Further addition of water molecules
resulted in no change around the metal ion. The addi-
tional water molecules occupied positions close to the
carboxylate C@O, implying hydrogen bonding. Based
on this result, we propose distorted trigonal bipyramidal
coordination for the metal ion with the carboxyl groups
in the equatorial positions and the amino nitrogen and a
solvent molecule occupying the two axial positions.
Zn2+
0.8
0.6
0.4
0.2
0
Hg2+
Ca2+
Mg2+
Co2+
Cd2+
2
Ni2+
Cu2+
450
300
350
400
500
Wavelength (nm)
Figure 2. Fluorescence spectrum of 2 in the presence of various metal
ions in 0.1 M HEPES buffer at pH 7 (kex = 300 nm).
trum of sensor 1 or 2 did not show any detectable
change in the presence of the metal ions.
Based on the results of the AM1 calculations, we
decided to investigate the effect of the number of acetyl
groups on the sensor. We synthesized compounds 2 and
3 (Chart 1) by controlling the amount of ethyl bromo-
acetate using the same procedure as the one used for
the synthesis of 1 (see Supplementary data for the
detailed procedure). An alternative structure of 1–Zn
complex could involve one iminodiacetate group and
the methoxy group. To test this possibility, we synthe-
sized anisidine derivative 4, which should be virtually
indistinguishable from 1 as a sensor if indeed only one
iminodiacetate group and the methoxy group were
involved in binding the metal ion.
Sensors 1 and 2 act as sensors due to enhanced fluores-
cence intensity rather than a change in the emission
wavelengths. The emission profile of the sensors as well
as their metal bound complexes have the same emission
profile. This implies that the fluorescence quantum yield
of the sensor is increased only in the presence of Zn(II)
ions. While the reason for this enhancement is still not
clear, it is possible that Zn(II) binding provides rigidity
to the molecule thereby blocking some of the nonradia-
tive decay pathways leading to an enhanced fluorescence
intensity.
Figure 3 compares the fluorescence intensity of sensors 1
and 2 in the presence of Zn(II) and Cd(II) metal ions.
When excited at 300 nm in 0.1 M HEPES buffer at pH
7 in the presence of 0.135 M NaCl, both the sensors emit
with approximately the same intensity. The emission
maxima for 2 and its complexes with the metal ions
are blue shifted by about 10 nm. While addition of
Cd(II) marginally affects the emission spectrum of 2, a
fourfold enhancement of intensity was observed for 1.
The fluorescence spectra for compounds 2–4 were mea-
sured in 0.1 M HEPES buffer at pH 7 in the presence of
0.135 M NaCl using Fluorolog-3 spectrofluorometer.
Compound 4 has an emission maximum at 370 nm,
which is quenched in the presence of Zn(II) ion (Supple-
mentary data). This result shows that the binding behav-
ior of 4 is different from sensor 1. Based on this
observation, we conclude that the geometry optimiza-
tion of the AM1 calculations is at least partially correct
in predicting the involvement of the acetyl groups from
both the amino groups. Compound 3 did not fluoresce
either in the presence or absence of the metal ions. While
this does not rule out Zn(II) binding of 3, this com-
pound cannot act as a fluorescence sensor for the metal
1.2
2 + Zn2+
1
ion. The fluorescence spectrum of compound 2 (kex
=
0.8
0.6
300 nm, pH 7) was very interesting (Fig. 2). As expected,
similar to compound 1, Co(II), Ni(II), and Cu(II)
quenched the fluorescence of sensor 2. Also, Ca(II)
and Mg(II) had no effect on the fluorescence of the sen-
sor. Surprisingly, Cd(II) enhanced the fluorescence
intensity of the sensor marginally, while Zn(II) addition
resulted in nearly twenty-fivefold enhancement of the
fluorescence intensity. These results do not rule out the
binding of the metal ions to the sensors, but the fluores-
cence enhancement is observed only for Zn(II). The
fluorescence enhancement of the sensor in the presence
of Zn(II) was independent of pH between 6 and 7.6.
However, at higher pH the fluorescence intensity
dropped due to the precipitation of Zn(OH)2. UV spec-
1 + Zn2+
0.4
1 + Cd2+
2 + Cd2+
0.2
1
2
0
300
350
400
Wavelength (nm)
450
500
Figure 3. Comparison of the fluorescence spectra of 1 and 2 in the
presence of Zn(II) and Cd(II) ions in 0.1 M HEPES buffer at pH 7
(kex = 300 nm).

7318
J. N. Ngwendson, A. Banerjee / Tetrahedron Letters 48 (2007) 7316–7319
Addition of Zn(II) to the sensor solutions led to nearly
tenfold enhancement of the fluorescence intensity of 1,
whereas a twenty-fivefold enhancement was observed
for 2. Following the fluorescence intensity change at
378 nm during the titration of sensor 2 with increasing
concentrations of Zn(II), a Kd value of 8.5 2.5 lM
was obtained following the best fit of the quadratic
equation.8 This value is comparable to that of sensor 1
(3.9 1.8 lM),7 indicating that three acetyl groups are
involved in binding the metal ion as suggested by the
AM1 calculations. The Kd value for binding of 2 with
other metal ions could not be determined since the
fluorescence change observed for these metals was negli-
gible. The binding constants for 3 and 4 were not deter-
mined since they did not show any sensory activity.
studies are consistent with a 1:1 sensor–Zn complex for
both 1 and 2. While, the possibility of the formation of
symmetric polynuclear species of the type [Sensor–Zn]x,
where x = 2 or higher cannot be ruled out, evidence so
far points to a mononuclear complex at this time: only
the fluorescence intensity is enhanced when the Zn(II)
ion is added, without any shift in the emission or
absorption spectra; and the HRMS (ESI) of the 2–Zn
complex gives
a
mass of 411.0122 (calculated:
411.0146). The HRMS data points toward a mononu-
clear species, however, the possibility of the observed
peak being a daughter ion rather than a molecular ion
peak cannot be ruled out. All attempts to recrystallize
the sensor–Zn complexes in an attempt to elucidate its
structure by X-ray diffraction resulted in the formation
of a white powder.
In biological systems, Ca(II) are present in higher con-
centrations than Zn(II) ions.6c,9 We incubated the sensor
with 10 equiv of Ca(II) at room temperature. This
resulted in a marginal enhancement of the fluorescence
intensity of the sensor (Fig. 4). When 1 equiv of Zn(II)
was added to the mixture, a fourfold enhancement of
fluorescence intensity at 375 nm was observed. This
result implies that there is at least a partial displacement
of Ca(II) ions by Zn(II) ions even in the presence of a
large excess of the Ca(II) ions. Similar displacements
of other metal ions with Zn(II) ion resulting in an
enhanced fluorescence intensity was observed for sen-
sors 1 and 2. Results of competitive binding experiments
will be reported in the future.
Of the four compounds studied, only 1 and 2 can act as
sensors. The number of acetyl group in these compounds
play an important role in determining the sensitivity and
selectivity of the metal ion. Our results indicate that two
acetyl groups must be present on at least one of the nitro-
gen atoms in order for the compound to fluoresce (1, 2,
and 4 fluoresced, while 3 did not). Also, at least three
acetyl groups must be present for the compound to act
as a Zn(II) ion sensor. Theoretical studies and the results
presented here suggests that all four acetyl groups do not
participate in binding Zn(II) at the same time, thereby
introducing fluxionality in the sensor–metal complex,
which results in lower sensitivity and selectivity of the
sensor. Three acetyl groups in 2 lead to a more rigid sen-
sor–metal complex leading to higher sensitivity and
selectivity without compromising the binding affinity.
The results imply that three acetyl groups are more effec-
tive than four acetyl groups in selective sensing of Zn(II)
ions. The selectivity of Zn(II) over Cd(II) and other
metal ions makes compound 2 a rare and important class
of fluorescent Zn(II) ion sensor.
Proton NMR of 1–Zn complex shows a deshielding
effect and peak broadening of all the protons relative
to the free sensor.7 A similar deshielding effect was
observed for sensor 2 when bound to Zn(II) though it
was much more prominent for aromatic protons. While
the deshielding effect for 1 was about 0.05 ppm, the two
aromatic protons of 2 were deshielded by about 0.44 and
0.64 ppm upon complexation with Zn(II). Moreover,
the peak broadening effect that was observed for 1 was
nonexistent in case of 2–Zn complex. This implies that
unlike 1–Zn complex, 2–Zn complex is not fluxional.
AM1 calculations, NMR spectra, and binding constant
Acknowledgments
Authors thank the University of North Dakota for fund-
ing this research. The authors also thank Dr. D.K. Sri-
vastava and Dr. David Pierce for helpful suggestions,
Dr. Julia Zhao for the use of fluorimeter, and Dr. Alena
Kubatova for the HRMS data (the funding for the spec-
trometer was provided by NSF CHE-0216038).
1.2
2 + 10 eq. Ca2+ + 1 eq. Zn2+
1
0.8
0.6
0.4
Supplementary data
Experimental procedures, fluorescence spectrum of 4,
determination of Kd for 2, and spectral data for all the
materials. Supplementary data associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.tetlet.2007.08.040.
2 + 10 eq. Ca2+
0.2
2
0
300
350
400
450
500
Wavelength (nm)
References and notes
Figure 4. Fluorescence spectra of 2 in the presence of 10 equiv of
Ca(II) ions before and after the addition of 1 equiv Zn(II) in 0.1 M
HEPES buffer at pH 7 (kex = 300 nm).
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