Design of a zinc(II) ion specific fluorescence sensor

Article abstract of DOI:10.1016/j.tetlet.2006.02.029

We report the synthesis of {[3-(biscarboxymethylamino)-2-methoxy-5-methylphenyl]carboxymethylamino}acetic acid, which functions as a Zn²⁺ selective fluorescence probe (sensor).

Full text of DOI:10.1016/j.tetlet.2006.02.029

Tetrahedron Letters 47 (2006) 2327–2330
Design of a zinc(II) ion specific fluorescence sensor
Julius N. Ngwendson,a Carrie L. Amiot,a D. K. Srivastavab and Anamitro Banerjeea,*
aDepartment of Chemistry, University of North Dakota, Grand Forks, ND 58202-9024, USA
bDepartment of Chemistry, Biochemistry and Molecular Biology, North Dakota State University, Fargo, ND 58102, USA
Received 30 December 2005; revised 30 January 2006; accepted 2 February 2006
Available online 21 February 2006
Abstract—We report the synthesis of {[3-(biscarboxymethylamino)-2-methoxy-5-methylphenyl]carboxymethylamino}acetic acid,
which functions as a Zn2+ selective fluorescence probe (sensor).
Ó 2006 Elsevier Ltd. All rights reserved.
In recent years, there has been a growing need for devel-
oping highly sensitive and selective probes for the detec-
tion of metal ions in biological and environmental
samples. A variety of divalent metal ions are known to
be involved in the structural, catalytic, and regulatory
aspects of the biological system, and some such metal
ions serve as prognostics of certain human diseases.1
For example, Cu2+, Zn2+, and Fe2+ have been found
to be involved in aggregating b-amyloid peptides during
the onset of Alzheimer’s disease.2 However, due to the
lack of metal ion specific probes, the relative contribu-
tion of one type of metal ion versus the other in causing
the disease is not clearly understood. The inability to
differentiate among different types of divalent metal ions
in biological samples has been one of the major impedi-
ments in the area of bio-analytical chemistry.
such compounds also exhibit weak selectivity
for Cu2+
encoded by facile changes in the coordination state
.
The origin of such selectivity appears to be
3d
of Zn2+ versus Cu2+
.
5
Due to diversity in functional roles of Zn2+ in biological
system (viz., DNA synthesis, apoptosis, structural
motifs in proteins, enzyme co-factors, etc.), we became
interested in designing the Zn2+ selective fluorescent
probes. In this endeavor, we noted that Cai et al.6
synthesized N,N,N0,N0-tetrakis(carboxylatemethyl)-2,6-
diaminocresol as the divalent metal binding probe, and
demonstrated that it forms five coordinate trigonal
bipyramidal structures with both Co2+ and Cu2+, and
the ligand–metal conjugate yields a charge-transfer band
around 300 nm. The structural data revealed that
besides carboxyl and amino groups, the phenolate oxy-
gen of the above ligand was involved in the coordination
bond with Co2+ and Cu2+, and that the latter exhibited
a somewhat distorted configuration. On assumption
that the phenolate oxygen might have some role in alter-
ing the coordination geometry of divalent metal ions, we
synthesized {[3-(biscarboxymethylamino)-2-methoxy-5-
methylphenyl]carboxymethylamino}acetic acid (com-
pound 1) in which the phenolic oxygen was methylated.
As elaborated below, the resultant compound emerged
out to be a Zn2+-specific fluorescent probe.
Although there has been some success in detection of
biologically significant metal ions by developing fluores-
cence probes (e.g., fura-2 for Ca2+), most of the probes
exhibit cross-reactivities for other metal ions.3 This is
not surprising since both physical and electronic proper-
ties of these metal ions are not too disparate, and they
tend to exhibit comparable binding affinities with their
cognate chelating agents. Consequently, not only syn-
thetic (organic) probes but also enzymatic probes exhibit
cross-reactivities among metal ions.4 Presently, quino-
line–sulfonamide containing compounds (and their
derivatives) are regarded to be as the ‘gold’ standards
for detecting fairly low concentrations of Zn2+, albeit
Scheme 1 describes the synthesis of compound 1 in three
simple steps. Commercially available 2-methoxy-5-
methyl-1,3-dinitrobenzene was reduced with tin and
concentrated HCl and neutralized with NH4OH to form
the diamine in 70% yield. The diamine was then N-alkyl-
ated with bromoethyl acetate in the presence of KI and
K2HPO4 to form compound 10 in 52% yield. Compound
Keywords: Zinc sensors; Fluorescence sensors.
*
Corresponding author. Tel.: +1 701 777 3941; fax: +1 701 777
2331; e-mail: abanerjee@chem.und.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.029

2328
J. N. Ngwendson et al. / Tetrahedron Letters 47 (2006) 2327–2330
OCH OCH
3
3
KI, K HPO
4
1. Sn/HCl
2
2
O N
NO
H N
2
NH
2
2
2
BrCH CO Et
2. NH OH
2
4
+
-
- +
EtO C
CO Et
K O C
CO K
2
2
2
2
OCH
OCH
3
3
+
-
- +
EtO C
N
N
CO Et
K O C
N
N
CO K
2
2
2
2
KOH
1'
1
Scheme 1. Synthesis of the sensor.
10 was hydrolyzed with KOH to form compound 1 (sen-
sor) in 96% yield.
enhancement of compound 1 was maintained even in the
presence of 100-fold excess of Ca2+ (data not shown).
Hence, compound 1 can be utilized to detect Zn2+ in
the physiological milieu containing the high concen-
trations of Ca2+. A cumulative account of these results
leads us to propose that compound 1 functions as a
Zn2+ specific fluorescent probe (sensor).
We determined the influence of divalent metal ions on
the fluorescence spectral profile of compound 1. Figure
1 shows the fluorescence emission spectra of 200 lM
compound 1 (kex = 300 nm) with nearly stoichiometric
concentrations of selected divalent metal ions (present
in the biological system). Note that compound 1 has a
weak fluorescence emission peak around 386–390 nm,
which is differently affected by different metal ions.
For example, whereas the emission intensity of com-
pound 1 (at 390 nm) is barely affected in the presence
of Mg2+, it is slightly increased in the presence of
Ca2+. On the contrary, the fluorescence intensity of
To determine the magnitude of Zn2+ induced fluores-
cence spectral changes of compound 1 as well as its
binding affinity, we performed a detailed spectrofluoro-
metric titration studies. Figure 2 (right panel) shows the
fluorescence emission spectra of compound 1 (corrected
for the buffer) as a function of increasing concentrations
of ZnCl2. The fluorescence emission intensity at 386 nm
(kex = 300 nm) shows a saturating profile as a function
of ZnCl2 (Fig. 2, left panel). Since the concentration of
compound 1 was comparable to the initial concentra-
tions of Zn2+, the binding constant of compound 1–
Zn2+ complex was calculated by a complete solution
of the quadratic equation, describing their interaction.7
The solid line is the best fit of the experimental data
for the Kd value of 3.9 1.8 lM and the stoichiometry
of 1:1 (i.e., 1 mol of bound Zn2+ per mol of compound
1). Of these parameters, the stoichiometry of the com-
plex suggested that only one of the two metal chelating
group was involved in the binding of Zn2+, the feature
compound 1 decreases in the presence of Cu2+, Ni2+
and Co2+. The most dramatic effect of fluorescence pro-
file of compound 1 is observed in the presence of Zn2+
,
.
As noteworthy from the data of Figure 1, the presence
of stoichiometric concentration of Zn2+, the fluores-
cence emission intensity of compound 1 is increased by
about 10-fold. Such an enhancement in fluorescence
intensity is not kinetically controlled as the time depen-
dent incubation of Zn2+ with compound 1 does not alter
the emission intensity. This deduction is equally valid
for the interaction of other metal ions with compound
1, irrespective of their spectral modulating features.
We further observed that the Zn2+-induced fluorescence
contrary to that observed for the binding of Co2+
/
Cu2+ with N,N,N0,N0-tetrakis(carboxylatemethyl)-2,6-
diaminocresol.6 We surmise that the origin of the un-
usual stoichiometry of compound 1–Zn2+ conjugate lies
in the methylation of phenolate oxygen of compound 1.
X 106
5
The question arises why compound 1 (vis a vis analo-
Zn2+
4
gous compounds reported in the literature) functions
as Zn2+ selective fluorescent sensor. Although
a
3
definitive answer to this question must await structural
determinations of different metal ions conjugated to
compound 1, circumstantial evidence leads us to conjec-
ture the following. It has been well established that
unlike Cu2+, Co2+, and Ni2+ (which predominate either
as the square planar or tetrahedral coordination state),
Zn2+ preferentially exists in the octahedral state, and
in this state, it can interact with all six groups contri-
buted by the two iminodiacetate moieties of compound
1. This hypothesis is supported by the stoichiometry of
compound 1–Zn2+ complex being equal to 1:1 (Fig. 2).
It is possible that compound 1–Zn2+ complex has limi-
ted nonradiative decay pathways available to it (leading
2
Ca2+
Mg2+
1
1
Co2+
Ni2+ Cu2+
0
300
320 340
360
380
400
420
440
460
480
500
Wavelength (nm)
Figure 1. Fluorescence emission spectra of compound 1 in 20 mM
HEPES buffer (pH 7.0) in the presence of different metal ions. The
concentrations of compound 1 and the metal ions were maintained at
200 lM.

J. N. Ngwendson et al. / Tetrahedron Letters 47 (2006) 2327–2330
2329
X 106
3
X106
3.2
Zn2+
2
1
0
2.4
1.6
0.8
0
K
= 3.9 1.8 µM
d
0
0.08
0.24
0.16
[Zn2+
tot (nM)
0.32
320
360
300
340
380
400
420
440
460
480
500
Wavelength (nm)
]
Figure 2. Fluorescence emission spectra of 100 lM of compound 1 in HEPES buffer in the presence of different concentrations of Zn2+. The
concentration of Zn2+ was increased from 10 to 300 lM (left panel). The right panel shows the increase in fluorescence intensity (at 386 nm) as a
function of Zn2+ concentration. The solid smooth line is the best fit of the data for the Kd value of 3.9 1.8 lM and the stoichiometry of 1:1 for
compound 1–Zn2+ complex.
Figure 3. 1H NMR trace of compound 1 (bottom trace) and the 1:1 mixture of 1 and Zn(II) (top trace) in D2O.
to enhanced fluorescence intensity) compared to com-
materials. This is a major advantage over the synthetic
protocols of known divalent metal ions sensors in the lit-
erature. Due to its high solubility in the aqueous medium,
it has the potential to serve as a selective fluorescence
probe (sensor) for detection of Zn2+ in biological sam-
ples. Moreover, low solubility of the complex could be
useful in extracting Zn2+ ions from environmental
matrix, when high concentrations of Zn are present. We
are currently in the process of modifying compound 1
to shift its excitation and emission spectra in the visible
region, as well as to enhance the binding affinity for
Zn2+, and we will report these findings subsequently.
pound 1 itself, while the tetracoordinated metal com-
plexes with unfilled d-orbitals (Cu2+, Co2+, and Ni2+
)
quench the excited singlet state of the sensor thereby
reducing its fluorescence intensity.
Compound 1–Zn complex has low solubility in water
(the solubility was not a factor in the concentrations
used in fluorescence experiments) and methanol. All
attempts at recrystallization of the complex so far led
to the formation of a white powder. Saturated solution
of the complex in D2O was subjected to 1H NMR
analysis in an attempt to elucidate the structure of the
1
complex. Comparison of the H NMR of compound 1
and its 1:1 mixture with Zn(II) (Fig. 3) indicated that
the zinc binding induces a deshielding effect on all the
protons accompanied by substantial peak broadening.
The broadening of the peaks imply that the complex is
fluxional, which may explain the poor recrystallization
properties.
Acknowledgements
The authors thank the ND-EPSCoR ‘Network in Cata-
lysis’ program (NSF-EPS-0132289), NIH (HL077201),
and the University of North Dakota, for funding this re-
search. We are grateful to Professor Ueyama Narikazu,
Mr. Kanamori Daisuke (both Osaka University),
Professor Harmon Abrahamson (University of North
Dakota), for helpful suggestions, and Professor Julia
It should be emphasized that compound 1 can be easily
synthesized in three steps from readily available starting

2330
J. N. Ngwendson et al. / Tetrahedron Letters 47 (2006) 2327–2330
3. (a) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1996,
118, 3053–3054; (b) Reany, O.; Gunnlaugsson, T.; Parker,
D. J. Chem. Soc., Perkin Trans. 2 2000, 1819–1831; (c)
Gunnlaugsson, T.; Lee, T. C.; Parkesh, R. Org. Lett. 2003,
5, 4065–4068; (d) Snitsarev, V.; Budde, T.; Stricker, T. P.;
Cox, J. M.; Krupa, D. J.; Geng, L.; Kay, A. R. Biophys. J.
2001, 80, 1538–1546; (e) Nolan, E. M.; Burdette, S. C.;
Harvey, J. H.; Hilderbrand, S. A.; Lippard, S. J. Inorg.
Chem. 2004, 43, 2624–2635, and references cited therein; (f)
Nolan, E. M.; Jaworski, J.; Okamoto, K.-I.; Hayashi, Y.;
Sheng, M.; Lippard, S. J. J. Am. Chem. Soc. 2005, 127,
16812–16823.
Xiaojun Zhao, for the use of the spectrofluorometer.
C.L.A. was also supported by GAANN fellowship (Uni-
versity of North Dakota).
Supplementary data
Experimental procedures and spectral data for all
the materials are available as supplementary data. Sup-
plementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2006.02.029.
4. Fierke, C. A.; Thompson, R. B. Biometals 2001, 14, 205–
222.
5. Glusker, J. P.; Katz, A. K.; Bock, C. W. Rigaku J. 1999, 16,
8–16.
6. Cai, L.; Xie, W.; Mahmoud, H.; Han, Y.; Wink, D. J.; Li,
S.; O’Connor, C. J. Inorg. Chim. Acta 1997, 263, 231–245.
7. Qin, L.; Srivastava, D. K. Biochemistry 1998, 37, 3499–
3508.
References and notes
1. Eiichi, K.; Koike, T. Chem. Soc. Rev. 1998, 27, 179–184.
2. Bush, A. L. Trends Neurosci. 2003, 26, 207–214.