Zinc(II)-selective ratiometric fluorescent sensors based on inhibition of excited-state intramolecular proton transfer

Article abstract of DOI:10.1002/chem.200305299

To develop a zinc(II)-selective emission ratiometric probe suitable for biological applications, we explored the cation-induced inhibition of excited-state intramolecular proton transfer (ESIPT) with a series of 2-(2′-benzene-sulfonamidophenyl)benzimidazo

Full text of DOI:10.1002/chem.200305299

FULL PAPER
Zinc(ii)-Selective Ratiometric Fluorescent Sensors Based on Inhibition
of Excited-State Intramolecular Proton Transfer
Maged M. Henary, Yonggang Wu, and Christoph J. Fahrni*[a]
Abstract: To develop a zinc(ii)-selec-
tive emission ratiometric probe suitable
for biological applications, we explored
the cation-induced inhibition of excit-
ed-state intramolecular proton transfer
(ESIPT) with a series of 2-(2’-benzene-
sulfonamidophenyl)benzimidazole de-
rivatives. In the absence of ZnII at neu-
tral pH, the fluorophores undergo
ESIPT to yield a highly Stokes× shifted
emission from the proton-transfer tau-
tomer. Coordination of ZnII inhibits
the ESIPT process and yields a signifi-
cant hypsochromic shift of the fluores-
cence emission maximum. Whereas the
paramagnetic metal cations CuII, FeII,
NiII, CoII, and MnII result in fluores-
cence quenching, the emission response
is not altered by millimolar concentra-
tions of CaII or MgII, rendering the sen-
sors selective for ZnII among all biolog-
ically important metal cations. Due to
the modular architecture of the fluoro-
phore, the ZnII binding affinity can be
readily tuned by implementing simple
structural modifications. The synthe-
sized probes are suitable to gauge free
ZnII concentrations in the micromolar
to picomolar range under physiological
conditions.
Keywords: fluorescent probes
¥
ligand design ¥ proton transfer ¥
sensors ¥ zinc
Introduction
centration, but is independent of the probe concentration,
pathlength, or spectral sensitivity of the instrument.[4] This
approach was originally developed for the visualization of
calcium-ion fluxes in live cells,[5] and a number of ratiomet-
ric sensors are now commercially available.[6] More recently,
studies on various eukaryotic cell lines indicate the presence
of intracellular compartments containing weakly bound,
labile ZnII (presumably up to millimolar concentrations). To
study the dynamics of compartmentalized ZnII in live cells
quantitatively, a cell-permeable, emission ratiometric sensor
would be very beneficial. Most of the currently available
cell-permeable ZnII probes function only as fluorescence
switches and are not suitable for ratiometric measure-
The development of cation-selective fluorescence sensors
has been an important goal of coordination chemistry for
decades.[1] Fluorescence-based probes provide high sensitivi-
ty and are therefore particularly well suited for the visuali-
zation of metal cations in biological environments.[2] The
majority of fluorescence sensors function as cation-respon-
sive optical switches that translate the binding event into an
increase or decrease of the emission intensity.[3] Due to the
linear relationship between intensity and cation concentra-
tion, quantitative measurements are, in principle, possible;
however, the emission intensity depends also on the sensor
concentration, which is typically not known with sufficient
accuracy in biological applications. Because the sensor
enters the cell through passive diffusion across the plasma
membrane, the intracellular concentration may vary as a
function of incubation time, temperature, membrane perme-
ability, and variations in cell size.
22]
ments.[17 Furthermore, due to the high cost of laser equip-
ment, modern 3D-imaging tools, such as confocal and multi-
photon microscopy, depend on a single-excitation light
source. To take advantage of these new imaging techniques,
the sensor must provide a spectral shift of the peak emis-
sion; however, with the exception of IndoZin,[23] currently
available cell-permeable ratiometric ZnII sensors require
dual excitation and are therefore not suitable for these ap-
plications.[6,23,24,52]
Ratiometric probes that exhibit a spectral shift upon bind-
ing of the cation offer an elegant solution to this problem.
The ratio of the emission intensities at two excitation or
emission wavelengths varies as a function of the cation con-
To develop a ZnII-selective emission ratiometric probe, we
recently explored the photophysics of cation-induced inhibi-
tion of excited-state intramolecular proton transfer
(ESIPT).[25] Benzimidazole derivatives (TA) containing an
intramolecular hydrogen bond undergo ESIPT and yield a
highly Stokes× shifted emission from the proton-transfer tau-
[a] Dr. M. M. Henary, Y. Wu, Prof. Dr. C. J. Fahrni
School of Chemistry and Biochemistry
Georgia Institute of Technology
770 State Street, Atlanta, GA 30332 (U.S.A.)
Fax : (+1)404-894-2295
E-mail: fahrni@chemistry.gatech.edu
30]
tomer TB (Scheme 1).[26
If the acidity of the proton at-
*
3015
Chem. Eur. J. 2004, 10, 3015 3025
DOI: 10.1002/chem.200305299
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
similar acidity of sulfonamides
and phenols, coordination of
ZnII should also result in dis-
placement of the proton and in-
hibition of the ESIPT process
as observed for the phenol de-
rivative I. Hence, to evaluate
this fluorophore as a platform
for the design of ZnII-selective
ratiometric probes, we synthe-
sized a family of water soluble
2-(2’-benzenesulfonamidophen-
yl)benzimidazole
derivatives
(III) and studied their emission
responses towards biologically
important metal cations under
physiological conditions. Fur-
thermore, to explore the tuna-
bility of the ZnII affinity and se-
lectivity, we introduced various
Lewis base donors (L) in prox-
imity to the bidentate cation
binding site.
Scheme 1. Simplified Jablonski diagram illustrating the increase of the emission energy upon metal-cation-in-
duced inhibition of excited-state intramolecular proton transfer.
tached to X is sufficiently high, coordination of a metal
cation M2+ will remove this proton and disrupt the ESIPT
process. Since emission of the coordinated species TM*
occurs with normal Stokes× shift, a significant blueshift is ex-
pected. For example, the acidity of the phenol proton in
2-(2’-hydroxyphenyl)benzimidazole (I) is sufficiently high,
such that ZnII coordination in organic solvents does indeed
yield the expected blueshift of the peak emission.[25]
Results and Discussion
Synthesis: By using a modular approach, ligands 7 9 were
synthesized from precursor 5 (Scheme 2). Coupling of com-
mercially available amine 1 with sulfonyl chloride 2,[32] fol-
lowed by oxidation of the benzylic hydroxyl group with
manganese(iv) oxide provided aldehyde 3. Copper(ii)-medi-
ated coupling with 1,2-diamino-3-hydroxymethylbenzene[33]
(4) followed by oxidation with manganese(iv) oxide gave
key intermediate 5, which was readily converted to the sub-
stituted ligands 7a, 8a, and 9a by means of reductive amina-
tion with the corresponding amines. The unsubstituted de-
rivative 6a is principally accessible by oxidative coupling of
3 with 1,2-phenylenediamine; however, since 2-(2’-amino-
phenyl)benzimidazole is commercially available, ligand 6a is
better synthesized through condensation with sulfonyl chlo-
ride 2 in a single step. The water-soluble acids 6b, 7b, 8b,
and 9b were obtained by hydrolysis with lithium hydroxide
in aqueous methanol.
Protonation equilibria: The protonation equilibria of the
four benzimidazole ligands 6b, 7b, 8b, and 9b were investi-
gated by potentiometric and spectrophotometric titrations.
For the determination of the pKa values, a solution of each
ligand in water (0.5 1mm) was titrated with 0.1m KOH at a
constant ionic strength of 0.1m KCl (Figure 1). The mea-
sured potential data were converted to ꢀlog[H3O+] based
on the electrode potential (E0) and slope s, which were de-
termined by Gran×s method;[34] nonlinear least-squares fit
analysis[35] provided the corresponding pKa values (Table 1).
Ligand 6b exhibits two protonation steps with pKa =8.0
and 4.5, which can be readily assigned to the nitrogen atom
of sulfonamide and the carboxylic acid moiety, respectively.
The tertiary-amine substituent in ligand 7b yields an addi-
However, in aqueous solution the proton-transfer emis-
sion of 2-(2’-hydroxyphenyl)benzimidazole is surprisingly
weak, and the spectrum is dominated by a normal emission
band, which presumably originates from the trans-rotamer
that cannot undergo ESIPT.[25,28] In contrast, 2-(2’-tosylami-
nophenyl)benzimidazole (II) lacks the electron lone pair re-
quired to stabilize the trans-rotamer by means of intramo-
lecular hydrogen bonding. With only the cis-rotamer pres-
ent, both in organic solvents and aqueous solution, the mol-
ecule exclusively exhibits the emission of the ESIPT tauto-
mer with a large quantum yield (0.3 0.5).[31] Given the
3016
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3015 3025

Zinc(ii)-Selective Sensors
3015 3025
Scheme 2. a) pyridine, 08C; b) MnO2, CH2Cl2, RT (53%, from 1); c) Cu(OAc)2, EtOH, reflux (20%); d) Cu(OAc)2, EtOH, reflux (36%); e) MnO2,
CH2Cl2, RT (86%); f) NaBH(OAc)3, Et2NH, 1,2-EtCl2 (76%); g) NaBH(OAc)3, N-2-picolyl-N-methylamine, 1,2-EtCl2 (97%); h) NaBH(OAc)3, N,N-
2,2’-dipicolylamine, 1,2-EtCl2 (80%).
(pKa =3.73) compared with 6b. The picolyl susbtituents in
8b and 9b substantially lower the pKa of the nitrogen atom
of the tertiary amine, an observation which is in agreement
with the reported low pKa (7.3) of bispicolylamine.[36,37] Be-
cause protonation of the nitrogen atom of sulfonamide is ex-
pected to occur in the same pH range, the pKa assignment
to either functional group is ambiguous for 8b and 9b.
To clarify this ambiguity we performed additional spectro-
photometric titrations. The spectral changes associated with
deprotonation of the nitrogen atom of sulfonamide provide
a characteristic signature to unequivocally assign the corre-
sponding pKa values. Deconvolution analysis of the UV-visi-
ble traces with the SPECFIT software package[38] provided
Figure 1. Potentiometric titration curves for ligands 6b 9b in water (0.1m
the absorption spectra for the species with protonated and
KCl, 258C; ligand concentrations (mm): 6b 1.0; 7b 0.47; 8b 0.64; 9b
deprotonated sulfonamide nitrogen atoms (Figure 2, left).
0.76).
The absorption maxima of the deconvoluted spectra are in-
cluded in Table 1 for ligands 6b, 7b, 8b, and 9b. Hence, for
tional protonation step with pKa =10.1. Consistent with the
presence of an ammonium cation, protonation of the car-
boxylic acid moiety now requires more acidic conditions
both ligands 8b and 9b the highest pKa values correspond
to protonation of the nitrogen atom of sulfonamide, while
the next lowest pKa values are associated with the picolyl-
3017
Chem. Eur. J. 2004, 10, 3015 3025
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

C. J. Fahrni et al.
FULL PAPER
Table 1. Protonation constants and photophysical data for ligands 6b, 7b, 8b, and 9b in aqueous solution.[a]
amine substituents. In summary,
deprotonation of the sulfon-
amide moiety occurs for all li-
gands at ꢀlog[H3O+]>8 and
yields qualitatively identical ab-
sorption spectra with a sharp
peak around 300 nm and a red-
shifted band centered around
330 nm (Figure 2, left). The
substituents attached to the
benzimidazole ring in ligand
7b, 8b, and 9b are electronical-
ly decoupled from the chromo-
phoric p-system and conse-
quently show no significant in-
fluence on the absorption spec-
tra. As illustrated by the distri-
bution diagram in Figure 3, the
protonated sulfonamide species
Species/Equilibrium
Data
6b
7b
8b
9b
[LHꢀ]/[H+][L2ꢀ
]
pK1
pK2
pK3
pK4
8.04ꢁ0.0310.12
4.50ꢁ0.038.02
n/a
ꢁ0.038.42
ꢁ10.037.3
ꢁ0.038.73
ꢁ0.036.49
ꢁ0.03
ꢁ0.03
[LH2]/[H+][LHꢀ]
[LH3+]/[H+][LH2]
[LH42+]/[H+][LH3
L2ꢀ
3.73ꢁ0.05
n/a
3.33ꢁ0.05
4.05ꢁ0.03
2.84ꢁ0.05
304 (12320)
332 (9220)
297
+
]
n/a
n/a
absorption lmax [nm][b]
301 (15870)
329 (10920)
296
418
6470
299 (12300)
300
460
11700
0.230.3
304 (10120)[f]
326 (7470)[f]
299[f]
303(15170)
329 (10600)
297
excitation lmax [nm][c]
emmission lmax [nm][c]
419[f]
419
419
Stokes× shift [cmꢀ1
]
6810[f]
6530
305 (12850)
309
6250
305 (10100)
308
LHꢀ
absorption lmax [nm][b]
excitation lmax [nm][d]
emission lmax [nm][d]
306 (9360)[g]
309[g]
469[g]
471
460
Stokes× shift [cmꢀ1
quantum yield[e]
]
11360[g]
11550
0.28
11050
[g]
2
0.09
[a] 0.1m KCl, 258C; [b] From deconvolution analysis, molar extinction coefficient [Lmolꢀ1 cmꢀ1] in parenthe-
ses; [c] pH 11.0, 0.1m KCl; [d] pH 7.00, 10mm PIPES, 0.1m KCl; [e] Quinine sulfate in 1n H2SO4 as standard;
[f] Monoprotonated species LHꢀ; [g] Diprotonated species LH2.
Figure 3. Calculated species distribution diagrams for ligands a) 6b,
b) 7b, c) 8b, and d) 9b.
dominate the equilibrium composition at neutral pH. How-
ever, the lower protonation constants of ligands 6b and 7b
are responsible for the presence of approximately 10% of
the sulfonamide anion, thus shifting the optimal conditions
for ratiometric cation measurements for these two ligands to
a lower pH range.
Upon excitation at the peak-absorption wavelength, all
four ligands 6b 9b display an intense fluorescence emission
with a quantum yield range of 9 32%. At neutral pH, the
Figure 2. Comparison of the UV-visible absorption (left) and normalized
fluorescence emission spectra (right) of the species with protonated
(c) and deprotonated (b) sulfonamide nitrogen atom in aqueous
solution (0.1m KCl, 258C) for ligands a) 6b, b) 7b, c) 8b, and d) 9b. The
emission spectra were recorded at pH 7.2 (c) and 11.0 (b). The UV-
visible traces were obtained through deconvolution of a series of spectra
with pH range 7 10.
3018
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3015 3025

Zinc(ii)-Selective Sensors
3015 3025
emission spectrum of each ligand consists of a single band
centered uniformly around 460 nm (Figure 2, right). The
spectra are not influenced by the choice of excitation wave-
length, suggesting the presence of a single, emissive species
in the excited-state manifold. The large Stokes× shifts, aver-
aging approximately 11000 cmꢀ1, are characteristic for emis-
[31]
*
sion of the corresponding proton-transfer tautomer TB.
Deprotonation of the nitrogen atom of sulfonamide disrupts
the ESIPT process and results in a blueshifted, normal emis-
sion band of the sulfonamide anion around 419 nm. Unlike
the structurally related 2-(2’-hydroxyphenyl)benzimidazoles,
which exhibit multiple emission bands under identical condi-
tions,[22,28] ligands 6b 9b display uniform photophysical
properties, which render them potentially suitable as cation-
selective probes.
Ground-state tautomerism: The NH-proton in benzimida-
zoles is known to undergo rapid, annular tautomerization.[39]
While in the case of ligands 6a/b the annular tautomeriza-
tion corresponds to a degenerate equilibrium, the tautomer
pairs of the substituted ligands 7a/b 9a/b are structurally
different and represent a nondegenerate, prototropic equi-
Figure 4. Variable-temperature 1H NMR spectra of ligand 7a in CD3OD
(3mm).
Complexation studies: Among the biologically important
metal cations, only ZnII yielded a bright, blueshifted fluores-
cence emission when added to an aqueous solution of any of
the four ligands 6b 9b. At neutral pH, coordination of CaII
and MgII is presumably too weak to replace the sulfonamide
proton, and gives no visible change of the fluorescence emis-
sion. The paramagnetic transition-metal cations MnII, CoII,
FeII, NiII, and CuII coordinate to the ligands, but partially or
completely quench the fluorescence emission. Given the se-
lective fluorescence response towards ZnII, we investigated
the interaction of this cation with each of the four ligands
6b 9b in more detail. A compilation of all thermodynamic
and photophysical data obtained from these studies is given
in Table 2.
1
librium (Scheme 3). The H NMR spectra of ligands 6a/b
Scheme 3. Nondegenerate prototropic equilibrium of the tautomer pairs
of the substituted ligands 7a/b 9a/b.
Table 2. Thermodynamic and photophysical data for the complexation of
ligands 6b 9b with ZnII in aqueous solution.[a]
9a/b all contain a single set of resonances for the benzimida-
zole protons. Conclusively, the tautomerization rate at room
temperature is too fast to be observed on the NMR time-
scale, resulting in a spectrum at the fast-exchange limit with
averaged signals. As exemplified by ligand 7a in CD3OD, at
lower temperatures the proton resonances undergo line
broadening (Figure 4); however, even at ꢀ608C the tauto-
merization rate does not reach the slow-exchange limit that
would provide the chemical-shift information for each tauto-
mer and their concentration ratio. The data set is therefore
not suitable to determine the equilibrium constant and acti-
vation barrier of the prototropic equilibrium.
Data
6b
7b
8b
9b
logK[b]
4.50ꢁ0.04 3.09ꢁ0.04 9.23ꢁ0.04 12.10ꢁ0.04
absorption lmax [nm][c]
excitation lmax [nm]
emission lmax [nm]
327
294
406
5950
0.44
8.46
19
318
299
297
300
415
9570
0.24
7.34
30
334
338
406
5310
0.14
11.6
82
420
Stokes× shift [cmꢀ1
Rmin F(400/500)[d]
Rmax F(400/500)[d]
Rmax/Rmin
]
7640
0.15
3.06
20
quantum yield[e]
0.22
0.230.17
0.21
[a] 0.1m KClO4, 258C, 10mm PIPES, pH 7.20; [b] Apparent binding con-
stant at pH 7.20; [c] From deconvolution analysis; [d] Ratio R of fluores-
cence emission intensities at 400 and 500 nm; [e] Quinine sulfate in 1n
H2SO4 as standard.
The tautomerization equilibrium constants for 4(7)-substi-
tuted benzimidazoles reported in the literature are very
close to 1, indicating near equivalence in the stability of the
N1(H) and N3(H) tautomers.[40] Thus, the stability difference
between the annular tautomers of ligands 6 9 is primarily
controlled by steric factors and presumably rather small. We
therefore assume that both tautomers are present in signifi-
cant concentrations, and that the previously discussed ab-
sorption and emission spectra correspond to the averaged
spectra of both tautomers.
Absorption and emission spectra: Since coordination to the
sulfonamide group is accompanied by loss of the nitrogen-
bound proton, the UV-visible spectrum should display a sim-
ilar redshift as previously observed for the deprotonated sul-
fonamide anion (Figure 2). Addition of ZnII triflate to
ligand 6b at neutral pH did indeed yield a new redshifted
band at 327 nm and a single isosbestic point at 318 nm (Fig-
3019
Chem. Eur. J. 2004, 10, 3015 3025
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

C. J. Fahrni et al.
FULL PAPER
ure 5a, left). When excited at this wavelength, the emission
spectra at various ZnII concentrations also revealed an iso-
sbestic point at 428 nm (Figure 5a, right), which is consistent
with a simple equilibrium system involving free and ZnII-
bound ligands. As expected, displacement of the sulfon-
amide proton by ZnII inhibits the ESIPT process and results
in a significant blueshift of the peak emission.
throughout the titration. Interestingly, the emission spectra
acquired under identical conditions revealed the same char-
acteristic blueshift as observed for 6b and 7b, and indicated
inhibition of the ESIPT process. This might initially appear
as a contradiction, however, the formation of the ESIPT
tautomer requires not only the presence of the sulfonamide
proton but also the proton-accepting electron lone pair of
the benzimidazole nitrogen atom. If this nitrogen atom is co-
ordinated to ZnII, the ESIPT process is inhibited regardless
of the protonation state of the sulfonamide group. This in-
terpretation is further supported by the apparent ZnII bind-
ing affinity of logK=9.2 (vide infra), which is significantly
higher than the apparent binding affinity of the picolylamine
moiety (logKZn =3.1, pH 7.2).[41]
Titration of ligand 9b with ZnII displayed a similar red-
shifted absorption band in the UV-visible spectrum as previ-
ously observed for 6b and 7b, indicating coordination of
ZnII to the sulfonamide nitrogen atom (Figure 5d, left). The
high apparent binding affinity of logK=12.1 at neutral pH
(vide infra) suggests formation of a pentadentate complex
involving coordination of all five nitrogen-donor atoms. This
assumption is additionally supported by a comparison with
the apparent ZnII affinity of dipicolylamine (logKZn =7.3at
pH 7.2),[41] which is five orders of magnitude lower than the
affinity of 9b. The fluorescence emission maxima again un-
dergo a blueshift (Figure 5d, right), which is in agreement
with the inhibition of ESIPT in favor of the normal emission
of the ZnII-bound fluorophore.
Apparent ZnII bindingaffinities : The binding affinities at
neutral pH of the four ligands 6b 9b were determined by
using nonlinear least-squares fit analysis of the emission re-
sponses at various ZnII concentrations (Table 2). For ligands
6b and 7b the ZnII affinities were directly obtained from
mol ratio plots with ZnII concentrations ranging between
10mm and 50mm. The two ligands (6b, 7b) bind ZnII with
logK=4.50ꢁ0.04 and 3.09ꢁ0.04, respectively, and form
both complexes with a 1:1 stoichiometry. Much to our sur-
prise, the diethylamino substituent attached to the benzimi-
dazole ring in 7b did not yield an increase, but rather a de-
crease in binding affinity. Since the UV-visible traces indi-
cate that the sulfonamide nitrogen atom is coordinated to
ZnII, it appears unlikely that the diethylamino group is also
involved in complexation to the metal center. Compared
with 8b and 9b, the nitrogen atom of the tertiary amine in
7b is approximately three orders of magnitude more basic,
and presumably remains protonated even in the presence of
millimolar concentrations of ZnII. The additional charge of
the resulting ammonium cation might therefore be responsi-
ble for the unexpectedly low ZnII affinity of 7b.
Initial mol ratio plots of the emission response of ligands
8b and 9b with ZnII revealed a fractional saturation of
>99% throughout the entire titration range, indicating a
significantly higher binding affinity compared with 6b or 7b.
Thus, to reliably measure the ZnII affinity of logK=9.23ꢁ
0.04 for 8b a set of ZnII/EGTA (EGTA=ethylenebis(oxy-
ethylenenitrilo)tetraacetic acid) buffer solutions were used
with free ZnII concentrations ranging between 10pm and
18nm. Similarly, the affinity of logK=12.10ꢁ0.04 for 9b
Figure 5. UV-visible absorbance (left) and fluorescence emission spectra
(right) of ligands a) 6b, b) 7b, c) 8b, and d) 9b as a function of added
ZnII (excitation at the isosbestic point of the UV-visible traces, pH 7.20,
10mm PIPES, 0.1m KClO4).
Ligand 7b shows qualitatively the same behavior as 6b
(Figure 5b); however, at ZnII concentrations above 10mm
the isosbestic point of the initial set of UV-visible traces is
no longer conserved, indicating the formation of a new spe-
cies (Figure 5b, left, dotted curves). The continuous increase
of the absorbance over the entire wavelength range contra-
dicts the presence of a stoichiometrically well-defined com-
plex; this is possibly a result of precipitation and/or the for-
mation of a coordination polymer.
In contrast, the UV-visible titration of ligand 8b with ZnII
did not yield the expected redshifted absorption band as ob-
served for 6b and 7b under identical conditions (Figure 5c,
left). Evidently, the sulfonamide nitrogen atom does not par-
ticipate in coordination to ZnII, and remains protonated
3020
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3015 3025

Zinc(ii)-Selective Sensors
3015 3025
was determined with a set of ZnII/HEDTA (HEDTA=N-
(2-hydroxyethyl)ethylenediaminetriacetic acid) buffer solu-
tions with free ZnII ranging between 0.16 13pm.
respectively. The lower affinity of ligand 7b does not signifi-
cantly expand the dynamic range relative to 6b, since ac-
cording to the previously discussed UV-visible data, the
binding stoichiometry is not conserved at ZnII concentra-
tions above 10mm (see absorption and emission spectra sec-
tion). This ligand was therefore excluded from the investiga-
tion of the emission response towards other biologically im-
portant metal cations.
Ratiometric measurements: If the peak excitation or emis-
sion wavelengths are shifted upon binding of the metal
cation, the ratio R of the intensities at two different wave-
lengths l1 and l2 is sufficient to determine the metal concen-
tration, regardless of the probe concentration, path length,
or other instrument-related parameters.[4] With a 1:1 binding
stoichiometry the ZnII concentration is given by Equa-
tion (1):
Selectivity towards ZnII: In coordination studies with other
biologically relevant metal cations, ligands 6b, 8b, and 9b
revealed a selective response towards ZnII. Since coordina-
tion of ZnII results in a blueshift of the emission maximum
to 405 nm, the emission intensity at this wavelength is a
good indicator for the selectivity of the probe response
(Figure 7). With up to millimolar concentrations of CaII and
ꢀ
ꢁ
Sapo
RmaxꢀR Sbnd
RꢀRmin
½ZnIIŠ ¼ Kd
ð1Þ
in which Rmin and Rmax are the limiting R values in the ab-
sence and at saturating levels of ZnII, respectively. The two
instrument-specific factors Sapo and Sbnd are determined from
solutions containing accurately known concentrations of the
probe and ZnII. Since Rmax/Rmin reflects the limiting dynamic
range and resolution for concentration measurements, this
parameter is a good measure to compare the performance
of various probes. For each of the four ligands 6b 9b, the
ratio of the emission intensities at 400 and 500 nm were de-
termined at various ZnII concentrations covering at least
one logarithmic unit above and below the corresponding dis-
sociation constants. Nonlinear least-squares fit analysis using
Equation (1) yielded the Rmin and Rmax values listed in
Table 2. The calculated Rmax/Rmin ratios range between 19 82
and compare well to the commercially available ratiometric
Figure 7. Emission intensity at 405 nm (lex =320 nm) of ligands 6b, 8b,
and 9b in response to various metal cations. Concentrations: 6b 10mm,
all M2+ 1mm; 8b 10mm, CaII, MgII 1mm, all other M2+ 10mm; 9b 10mm,
CaII, MgII 1mm, all other M2+ 10mm (pH 7.2, 10mm PIPES, 0.1m KClO4).
calcium probes indo-1 (Rmax/Rmin =21) or fura-2 (Rmax/Rmin
=
45).[4] A plot of the emission intensity ratio versus the free
ZnII concentration further illustrates the dynamic range of
each of the ligands 6b 9b (Figure 6). For a better compari-
son, Rmax/Rmin was normalized to unity and the experimental
ratios R were scaled accordingly. Because reliable concen-
tration measurements are only feasible between 20 80%
fractional saturation,[42] the analytically useful concentration
range is typically restricted to one order of magnitude above
and below the dissociation constant of the probe. Fluoro-
phore 6b is thus suitable to gauge free ZnII between 30mm
and 3mm, whereas the high-affinity probes 8b and 9b can
be used for concentrations between 1 10nm and 0.2 20pm,
MgII the emission spectra of all three ligands essentially re-
mained unchanged, whereas the paramagnetic metal cations
CuII, FeII, NiII, CoII, and MnII resulted in fluorescence
quenching. We also investigated the emission response for
CdII: a nonbiological d10 metal cation that often exhibits
similar coordination properties to ZnII. In the presence of
CdII all three ligands showed increased emission intensities
at 405 nm; however, only 8b yielded a similarly strong fluo-
rescence enhancement as observed with ZnII (Figure 7).
Figure 8 shows the emission intensity ratios at 400 and
500 nm as a function of selected divalent metal cations
(dark columns). With the exception of CuII, the emission re-
sponse of 6b was not significantly altered by millimolar con-
centrations of the tested cations. The ligands 8b and 9b re-
vealed a similar trend; however, CoII, NiII, and FeII also
yielded an increased emission ratio. Nevertheless, because
the fluorescence emission in the presence of these cations is
very weak, it should be possible to distinguish this ratio in-
crease from the ZnII-selective emission change (Figure 7).
To further gauge the selectivity of the ligands, the emis-
sion response of each cation was measured in the presence
of an equimolar amount of ZnII (Figure 8, light columns).
Under these conditions the emission ratios for 8b and 9b
were essentially restored to the same values as those ob-
served for the ZnII-saturated probe. Only the measurements
Figure 6. Change of the emission intensity ratio at 400 and 500 nm (lex
=
320 nm) for ligands 6b 9b as a function of free ZnII (pZn=ꢀlog[ZnIIfree],
pH 7.2, 10mm PIPES, 0.1m KClO4).
3021
Chem. Eur. J. 2004, 10, 3015 3025
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

C. J. Fahrni et al.
FULL PAPER
troscopically silent ligands. The high-affinity probes 8b and
9b should be particularly suitable for the measurement of
the binding affinities of ZnII metalloproteins, whose binding
constant is typically in the range between 108 1012 mꢀ1 (e.g.,
alkaline phosphatase, KZn =107 108,[43] carboxy peptidase,
K
Zn =2.1î108,[44] carbonic anhydrase, KZn =1.3î1012[45]). We
anticipate that simple structural modifications through intro-
duction of suitable substituents should yield a sufficiently
redshifted excitation maximum and also render the fluoro-
phore platform suitable for microscopy applications. To
evaluate their suitability as ZnII-selective probes in multi-
photon laser microscopy, we are currently exploring the
two-photon cross sections of these fluorophores. This ap-
proach would be particularly attractive for in vivo applica-
tions, since the required excitation wavelength corresponds
to one-half of the single-photon energy.
Experimental Section
Materials and reagents: 2-Aminobenzyl alcohol (Aldrich, 98%), 2-(2’-
aminophenyl)benzimidazole (Alfa-Aesar, 98%), phenol (Aldrich, 99%),
ethyl bromoacetate (Aldrich, 98%), manganese dioxide (Aldrich, 99%),
1,2-phenylenediamine (Aldrich, 99%), copper acetate monohydrate (Al-
drich, 98%), thionyl chloride (Aldrich, 99%), 2,2’-dipicolylamine (TCI,
97%), N,N-diisopropylethyl amine (Aldrich, 99%), N-(2-hydroxyethyl)-
ethylenediaminetriacetic acid (HEDTA, Aldrich, 99%), ethylenebis(oxy-
ethylenenitrilo)tetraacetic acid (EGTA, Aldrich, 97%). NMR: d in ppm
versus SiMe4 (0 ppm, 1H, 400 MHz). MS: selected peaks; m/z. Melting
points are uncorrected. Flash chromatography: Merck silica gel (240
400 mesh). TLC: 0.25 mm, Merck silica gel 60 F254, visualizing at 254 nm
or with 2% KMnO4 solution.
Figure 8. Emission intensity ratios at 400 and 500 nm in response to vari-
ous metal cations (pH 7.2, 10mm PIPES, 0.1m KClO4): a) 10mm 6b, all
M2+ 1mm; b) 10mm 8b, CaII, MgII 1mm, all other M2+ 10mm; c) 10mm so-
lution of 9b, CaII, MgII 1mm, all other M2+ 10mm. Binding competition
measurements were acquired after equilibration for 3h (6b) and 24h (8b
and 9b) with solutions containing equimolar concentrations of ZnII and
the respective metal cation.
with CuII resulted in a significant change in the emission
ratio, but again induced fluorescence quenching. In compari-
son, the response of ligand 6b, which is lacking any addi-
tional coordinating pyridine groups, was less selective.
Whereas CaII, MgII, and MnII did not interfere with ZnII
binding, the emission ratios for CoII, FeII, NiII, and CuII were
significantly lower suggesting competitive binding of these
cations; however, for CdII-induced fluorescence enhance-
ments the emission ratios were essentially restored in the
competition assays with all three ligands, indicating a lower
binding affinity of CdII compared with ZnII.
Synthesis: 2,3-Diaminobenzyl alcohol[33] 4 and ethyl 2-phenoxyacetate[46]
were synthesized following the published procedures.
Ethyl (4-chlorosulfonylphenoxy)acetate (2): Chlorosulfonic acid (7.0 mL)
was added dropwise to
a solution of ethyl 2-phenoxyacetate (2 g,
11 mmol) in dichloromethane (50 mL) and cooled with ice. After stirring
for 3h at RT, the reaction mixture was poured into ice-cold water
(20 mL) and extracted two times with diethyl ether (40 mL). The com-
bined organic extracts were dried with anhydrous MgSO4 and concentrat-
ed under reduced pressure to provide the sulfonyl chloride 2 as a yellow-
ish oil (2.40 g, 78% yield). 1H NMR (CDCl3, 400 MHz): d=1.32 (t, J=
7.1 Hz, 3H), 4.29 (q, J=7.1 Hz, 2H), 4.73(s, 2H), 7.04 (d, J=9.3Hz,
2H), 7.97 ppm (d, J=9.3Hz, 1H); MS (70 eV): m/z (%): 278/280 (39/15)
[M+], 243(100); EI-HRMS: m/z calcd (%) for [M+] C10H11ClO5S:
278.0016; found: 278.0049.
Conclusion
Ethyl [4-(2-formylphenylsulfamoyl)phenoxy]acetate (3): A mixture of 1
(500 mg, 4.06 mmol), 2 (1.4 g, 5.0 mmol), and pyridine (0.5 mL) in di-
chloromethane (5 mL) was stirred at RT for 2 h. The reaction mixture
was quenched with water (50 mL) and extracted twice with dichloro-
methane. The combined organic extracts were dried with anhydrous
MgSO4 and concentrated under reduced pressure to give ethyl [4-(2-hy-
droxymethylphenylsulfamoyl)phenoxy]acetate as a yellow oil (1.42 g).
Without further purification, the product was dissolved in dichlorome-
thane (10 mL) and stirred together with manganese dioxide (4.8 g) at RT
overnight. The suspension was filtered through a pad of Celite and con-
centrated under reduced pressure. Recrystallization from ethyl acetate
provided aldehyde 3 as a white solid (783mg, 53% yield). M.p. 83 85 8C;
1H NMR (CDCl3, 400 MHz): d=1.27 (t, J=7.1 Hz, 3H), 4.24 (q, J=
7.1 Hz, 2H), 4.62 (s, 2H), 6.89 (d, J=8.7 Hz, 2H), 7.15 (t, J=7.6 Hz,
1H), 7.49 (t, J=7.1 Hz, 1H), 7.58 (dd, J=7.4, 1.4 Hz, 1H), 7.66 (d, J=
8.2 Hz, 1H), 7.81 (d, J=8.7 Hz, 2H), 9.81 (s, 1H), 10.75 ppm (s, 1H); 13C
NMR (CDCl3, 100 MHz): d=14.6, 62.0, 65.6, 115.1, 118.0, 122.1, 123.2,
129.7, 132.2, 136.0, 136.3, 140.0, 161.5, 167.9, 195.0 ppm; MS (70 eV): m/z
(%): 363 (34) [M+], 243(35), 120 (100); EI-HRMS: m/z calcd for [M+]
C17H17NO6S: 363.0776; found: 363.0771; elemental analysis calcd (%) for
In summary, the benzimidazole derivatives 6b 9b demon-
strate that cation-induced inhibition of ESIPT leads to a
substantial spectral shift of the fluorescence emission and is
a promising concept for the development of ZnII-selective
ratiometric probes. The modular architecture of the probe
principally allows for tuning of the binding affinity as illus-
trated with the picolylamine-substituted derivatives 8b and
9b. Because the spectral characteristics are not significantly
altered by the attached substituents, the approach is suitable
for generating a family of probes that can be used with a
single filter set. Probes 6b 9b require excitation in the UV
range, which is a limiting factor for fluorescence microscopy
applications. However, the probes can be used in any cu-
vette-based application, for example, in competition experi-
ments for the determination of the binding affinities of spec-
3022
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3015 3025

Zinc(ii)-Selective Sensors
3015 3025
C17H17NO6S (363.4): C 56.19, H 4.72, N 3.85; found: C 56.26, H 4.64, N
3.93.
phase HPLC (10î300 mm C18 column, CH3CN/H2O (0.01%TFA)
75:25!98:2); M.p. 1408C (decomp); 1H NMR ([D6]DMSO, 400 MHz):
d=4.66 (s, 2H), 6.90 (d, J=8.8 Hz, 2H), 7.19 (t, J=7.7 Hz, 1H), 7.28
7.31 (m, 2H), 7.40 (t, J=7.7 Hz, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.65 7.67
(m, 4H), 8.02 (d, J=8.2 Hz, 1H), 13.23 ppm (s, 2H); MS (70 eV): m/z
(%): 423(20) [ M+], 300 (17), 208 (100); EI-HRMS: m/z calcd (%) for
[M+] C21H17N3O5S: 423.0889; found: 423.0863.
Ethyl {4-[2-(4-formyl-1H-benzimidazol-2-yl)phenylsulfamoyl]phenoxy}-
acetate (5): Acetic acid (0.280 mL), aldehyde 3 (1.11 g, 3.06 mmol) in
MeOH (5 mL), and copper(ii) acetate monohydrate (610 mg) in water
(10 mL) were sequentially added with stirring to a solution of 2,3-diami-
nobenzyl alcohol 4 (300 mg, 2.17 mmol) in EtOH/H2O (20 mL, 1:1). The
mixture was heated under reflux for 3h, filtered hot, and the residue
washed with water. The precipitate was redissolved in a mixture of etha-
nol (12 mL) and concd HCl (2.2 mL). After addition of Na2S¥9H2O
(1.05 g in 8 mL water) the mixture was heated under reflux for 1 h,
cooled to RT, and filtered through a pad of Celite to remove the precipi-
tated CuS. The filtrate was neutralized with aqueous NaHCO3 and ex-
tracted three times with dichloromethane. The combined organic extracts
were dried with MgSO4 and concentrated under reduced pressure. The
crude product was purified on silica gel (hexanes/ethyl acetate 1:2!1:1)
to provide the hydroxymethyl-substituted benzimidazole as a glassy, yel-
lowish solid (378 mg, 36% yield). M.p. 54 568C; 1H NMR (CDCl3,
400 MHz) (note: tautomeric proton exchange broadens a few signals):
d=1.30 (t, J=7.1 Hz, 3H), 4.26 (q, J=7.1 Hz, 2H), 4.50 (s, 2H), 5.02 (s,
2H), 6.39 (d, J=8.7 Hz, 2H), 7.09 (brs, 1H), 7.15 (t, J=7.6 Hz, 2H), 7.22
(d, J=7.6 Hz, 1H), 7.33 (brs, 1H), 7.32 (t, J=7.6 Hz, 2H), 7.49 (d, J=
7.6 Hz, 1H), 7.67 (brs, 1H), 7.73(d, J=7.6 Hz, 1H), 9.80 (brs, 1H),
9.99 ppm (brs, 1H); MS (70 eV): m/z (%): 481 (65) [M+], 435 (27), 312
(31), 220 (100); EI-HRMS: m/z calcd (%) for [M+] C24H23N3O6S:
481.1308; found: 481.1297.
Ethyl {4-{2-{4-[(diethylamino)methyl]-1H-benzimidazol-2-yl}phenylsulfa-
moyl}phenoxy}acetate (7a): Diethyl amine (31 mL, 0.292 mmol) and
sodium triacetoxyborohydride (66 mg, 0.313 mmol) were added to a solu-
tion of aldehyde 5 (100 mg, 0.209 mmol) in 1,2-dichloroethane (15 mL).
The reaction mixture was allowed to stir for 2.5 h under N2 and was then
quenched by the addition of 1m aqueous NH4OH (3mL). The product
was extracted twice with dichloromethane and the combined organic ex-
tracts were dried with MgSO4. The organic solvent was evaporated under
reduced pressure and the residue purified by using flash chromatography
on silica gel (dichloromethane/methanol/TFA 50:1:0.25!50:1:0.5) to pro-
vide ligand 7a as a glassy, pale yellow solid (85 mg, 76% yield). M.p. 54
568C; 1H NMR (CDCl3, 400 MHz): d=1.15 (t, J=7.1 Hz, 3H), 1.23 (t,
J=7.1 Hz, 6H), 2.68 (q, J=6.9 Hz, 4H), 4.01 (s, 2H), 4.20 (q, J=7.1 Hz,
2H), 4.52 (s, 2H), 6.70 (d, J=8.8 Hz, 2H), 7.08 7.13(m, 2H), 7.20 (t, J=
7.7 Hz, 1H), 7.30 (t, J=7.7 Hz, 1H), 7.30 (t, J=8.2 Hz, 1H), 7.63(brs,
1H), 7.67 (d, J=7.7 Hz, 1H), 7.70 7.74 (m, 3H), 9.44 (brs, 1H),
12.52 ppm (brs, 1H); 13C NMR (CDCl3, 100 MHz): d=8.1, 10.5, 43.2,
52.2, 57.9, 61.4, 110.6, 112.7, 114.6, 116.3, 118.9, 119.1, 119.6, 120.6, 122.3,
122.6, 125.4, 126.8, 128.6, 129.5, 134.0, 145.7, 156.8, 164.0 ppm; MS
(70 eV): m/z (%): 536 (1) [M+], 507 (52), 465 (100), 222 (100), 72 (25);
EI-HRMS: m/z calcd (%) for [M+] C28H32N4O5S: 536.2093; found:
536.2065; elemental analysis calcd (%) for C28H32N4O5S¥0.5H2O (545.7):
C 61.63, H 6.10, N 10.27; found: C 61.83, H 6.13, N 9.93.
The above intermediate (482 mg, 1.0 mmol) was dissolved in dichloro-
methane (15 mL) and stirred with manganese dioxide (1.1 g) at RT over-
night. The suspension was filtered through a pad of Celite and concen-
trated under reduced pressure. Recrystallization from diethylether pro-
vided aldehyde 5 as a glassy, yellow solid (415 mg, 86% yield). M.p. 65
678C; 1H NMR (CDCl3, 400 MHz): d=1.24 (t, J=7.1 Hz, 3H), 4.21 (q,
J=7.1 Hz, 2H), 4.52 (s, 2H), 6.65 (d, J=8.8 Hz, 2H), 7.17 (t, J=8.0 Hz,
1H), 7.39 (td, J=8.8, 1.7 Hz, 1H), 7.49 (t, J=8.0 Hz, 1H), 7.64 (d, J=
9.3Hz, 2H), 7.68 (dd, J=8.8, 1.1 Hz, 1H), 7.79 (dd, J=8.5, 1.7 Hz , 2H),
8.11 (d, J=7.7 Hz, 1H), 10.11 (s, 1H), 10.87 (brs, 1H), 12.35 ppm (s,
2H); 13C NMR (CDCl3, 100 MHz): d=14.5, 61.9, 65.3, 114.5, 115.6, 116.4,
121.0, 121.3, 123.0, 123.8, 126.2, 126.9, 127.8, 129.3, 131.7, 132.2, 138.0,
149.0, 151.9, 160.9, 167.9, 192.4 ppm; MS (70 eV): m/z (%): 479 (64) [M+
], 415 (25), 378 (5), 328 (50), 236 (100), 209 (11), 181 (9); EI-HRMS: m/z
calcd (%) for [M+] C24H21N3O6S: 479.1151; found: 479.1145; elemental
analysis calcd (%) for C24H21N3O6S (479.5): C 60.12, H 4.41, N 8.76;
found: C 60.24, H 4.49, N 8.66.
{4-{2-{4-[(Diethylamino)methyl]-1H-benzimidazol-2-yl}phenylsulfamoyl}-
phenoxy}acetic acid (7b): Ethyl ester 7a (22 mg, 0.041 mmol) was hydro-
lyzed as described for acid 6b to provide the free acid 7b (12 mg, 58%).
M.p. 225 2278C; 1H NMR (CD3OD, 400 MHz): d=1.42 (t, J=7.1 Hz,
6H), 3.35 (q, J=7.4 Hz, 4H), 3.63 (brs, 1H), 4.34 (s, 2H), 4.74 (s, 2H),
6.61 (d, J=8.2 Hz, 2H), 7.24 (t, J=7.4 Hz, 1H), 7.32 (d, J=7.7 Hz, 2H),
7.34 7.45 (m, 3H), 7.69 (t, J=6.9 Hz, 2H), 7.81 ppm (dd, J=8.7, 1.1 Hz,
1H); MS (70 eV): m/z (%): 509 (100) [M+1]+, 435 (5), 338 (15), 295
(44); ESI-TOF-HRMS: m/z calcd (%) for [M+1]+ C26H29N4O5S:
509.1859; found: 509.1884.
Ethyl {4-{2-{4-[(methylpyridin-2-ylmethylamino)methyl]-1H-benzimida-
zol-2-yl}phenylsulfamoyl}phenoxy}acetate (8a): Prepared as described for
7a by reductive amination of aldehyde 5 (100 mg, 0.209 mmol) with
methyl picolylamine[47] (100 mg, 0.819 mmol), providing 8a as a glassy,
pale yellow solid (119 mg, 97% yield). M.p. 56 588C; 1H NMR (CDCl3,
400 MHz): d=1.22 (t, J=7.1 Hz, 3H), 2.29 (s, 3H), 3.74 (s, 2H), 3.89 (s,
2H), 4.18 (q, J=7.1 Hz, 2H), 4.41 (s, 2H), 6.64 (m, 2H), 7.03(d, J=
7.1 Hz, 1H), 7.14 7.18 (m, 3H), 7.27 (d, J=7.7 Hz, 1H), 7.33 (td, J=7.7,
1.7 Hz, 1H), 7.65 (dd, J=7.7, 2.2 Hz, 1H), 7.68 7.73(m, 3H), 7.78 (dd,
J=8.2, 1.1 Hz, 1H), 8.18 (d, J=7.7 Hz, 1H), 8.43(d, J=4.4 Hz, 1H),
13.48 ppm (brs, 2H); 13C NMR (CDCl3, 100 MHz): d=14.4, 43.0, 58.8,
61.8, 63.1, 65.3, 114.5, 117.6, 118.2, 120.3, 122.1, 122.5, 122.8, 123.4, 123.6,
123.9, 127.2, 129.4, 130.5, 132.8, 133.1, 137.3, 138.0, 143.0, 149.2, 150.5,
159.0, 160.6, 167.9 ppm; MS (70 eV): m/z (%): 586 (100) [M+1]+, 3 44
(13), 293 (27); ESI-TOF-HRMS: m/z calcd (%) for [M+1]+
C31H32N5O5S: 586.2124; found: 586.2089; elemental analysis calcd (%)
for C31H31N5O5S (585.7): C 63.57, H 5.34, N 11.96; found: C 63.57, H
5.44, N 11.78.
Ethyl {4-[2-(1H-benzimidazol-2-yl)phenylsulfamoyl]phenoxy}acetate (6a):
A solution of 2-(2’-aminophenyl)benzimidazole (310 mg, 1.48 mmol) and
2 (500 mg, 1.79 mmol) in pyridine (3mL) was stirred for 2 h. The reac-
tion mixture was diluted with water (20 mL) and extracted twice with
ethyl acetate (40 mL). The combined organic extracts were dried with
MgSO4 and concentrated under reduced pressure. The crude product was
purified by flash chromatography on silica gel (hexanes/ethyl acetate 2:1)
to give ligand 6a as a yellowish solid (400 mg, 60% yield). M.p. 134
1
1368C; H NMR ([D6]DMSO, 400 MHz): d=1.12 (t, J=6.6 Hz, 3H), 4.08
(q, J=7.1 Hz, 2H), 4.77 (s, 2H), 6.92 (d, J=8.8 Hz, 2H), 7.19 (t, J=
7.7 Hz, 1H), 7.28 7.31 (m, 2H), 7.40 (t, J=7.7 Hz, 1H), 7.60 (d, J=
8.8 Hz, 1H), 7.64 7.67 (m, 4H), 8.02 (d, J=8.2 Hz, 1H), 13.23 ppm (s,
2H); 13C NMR (CDCl3, 100 MHz): d=14.8, 61.5, 65.4, 115.7, 116.4, 119.2,
123.8, 124.0, 128.0, 129.5, 131.5, 131.8, 151.0, 161.5, 168.5, 168.5 ppm; MS
(70 eV): m/z (%): 451.1 (44) [M+], 387.1 (14), 300.1 (40), 208.1 (100); EI-
HRMS: m/z calcd (%) for [M+] C23H21N3O5S: 451.1202; found:
451.1189; elemental analysis calcd (%) for C23H21N3O5S (451.5): C 61.18,
H 4.69, N 9.31; found: C 61.09, H 4.72, N 9.36.
{4-{2-{4-[(Methylpyridin-2-ylmethylamino)methyl]-1H-benzimidazol-2-
yl}phenylsulfamoyl}phenoxy}acetic acid (8b): Ethyl ester 8a (40 mg,
0.068 mmol) was hydrolyzed as described for acid 6b to give free acid 8b
(23 mg, 61%). M.p. 133 1358C; 1H NMR (CDCl3, 400 MHz): d=2.47 (s,
3H), 3.63 (brs, 1H), 4.08 (s, 2H), 4.22 (s, 2H), 4.36 (s, 2H), 6.40 (d, J=
8.3Hz, 2H), 7.06 7.18 (m, 4H), 7.28 (d, J=6.1 Hz, 2H), 7.31 (d, J=
7.7 Hz, 4H), 7.69 (d, J=6.7 Hz, 2H), 7.75 (d, J=7.1 Hz, 2H), 8.55 ppm
(d, J=4.4 Hz, 1H); MS (70 eV): m/z (%): 558 (100) [M+1]+, 344 (5),
279, (19); ESI-TOF-HRMS: m/z calcd (%) for [M+1]+ C29H28N5O5S:
558.1811; found: 558.1846.
{4-[2-(1H-Benzimidazol-2-yl)phenylsulfamoyl]phenoxy}acetic acid (6b):
Ethyl ester 6a (170 mg, 0.38 mmol) was added to
a solution of
LiOH¥H2O (350 mg) in a mixture of methanol (1 mL) and water (1 mL).
The mixture was stirred at RT for 1 h, and the organic solvent subse-
quently removed under reduced pressure. The aqueous residue was acidi-
fied by the addition of 1m HCl and the product then extracted twice with
ethyl acetate. The combined organic extracts were dried with MgSO4 and
concentrated under reduced pressure to provide acid 6b as a yellowish
solid (140 mg, 88% yield). For the photophysical studies a sample of the
compound (10 mg) was further purified by semipreparative reversed-
Ethyl {4-{2-{4-[(bispyridin-2-ylmethylamino)methyl]-1H-benzimidazol-2-
yl}phenylsulfamoyl}phenoxy}acetate (9a): Prepared as described for 7a
3023
Chem. Eur. J. 2004, 10, 3015 3025
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

C. J. Fahrni et al.
FULL PAPER
by reductive amination of aldehyde 5 (40 mg, 0.083mmol) with 2,2 ’-dipi-
colylamine (22 mg, 0.11 mmol), providing 9a as a glassy solid (44 mg,
80% yield). M.p. 65 678C; 1H NMR (CDCl3, 400 MHz): d=1.23(t, J=
7.1 Hz, 3H), 3.89 (s, 2H), 3.91 (s, 4H), 4.19 (q, J=7.1 Hz, 2H), 4.41 (s,
2H), 6.66 (d, J=9.3Hz, 2H), 7.09 7.20 (m, 4H), 7.25 (d, J=7.6 Hz, 2H),
7.36 (td, J=8.5, 1.4 Hz, 1H), 7.54 (td, J=7.6, 1.6 Hz, 2H), 7.72 7.77 (m,
3H), 7.81 (dd, J=8.2, 1.1 Hz, 1H), 8.17 (dd, J=7.9, 1.1 Hz, 1H), 8.40 (d,
J=3.8 Hz, 2H), 13.20 (brs, 2H), 14.11 ppm (s, 1H); 13C NMR (CDCl3,
400 MHz): d=14.5, 56.5, 59.9, 61.9, 65.4, 114.6, 118.6, 120.6, 122.3, 122.6,
123.0, 123.3, 123.6, 123.7, 126.9, 129.5, 130.6, 133.0, 133.3, 137.1, 138.1,
143.0, 149.0, 150.6, 159.2, 160.7, 167.9 ppm; MS (70 eV): m/z (%): 662 (5)
[M+], 570 (100), 465 (13), 222 (40), 198 (53), 93 (78); EI-HRMS: m/z
calcd (%) for [M+] C36H34N6O5S: 662.2311; found: 662.2276; elemental
analysis calcd (%) for C36H34N6O5S¥0.5H2O (671.8): C 64.37, H 5.25, N
12.51; found: C 64.76, H 5.13, N 12.16.
varied between 5 and 11. The emf of each solution was directly measured
in the UV quartz cell (electrode diameter 3mm) and converted to
ꢀlog[H3O+] using the E8 and slope, as obtained from the electrode cali-
bration procedure described above. The raw spectral and emf data were
processed with nonlinear least-squares fit analysis using the SPECFIT
software package.[38]
Complex stability constants: All reported stability constants K were
measured at pH 7.2 (PIPES 10 mm, 0.1m KClO4 ionic background) and
refer to apparent stability constants.[50]
Determination of the apparent ZnII affinity of ligands 6b and 7b: UV/Vis
spectrophotometric titrations were performed with
a 60mm solution
(3.0 mL cell volume) of the corresponding ligand in PIPES buffer
(10mm, pH 7.20, 0.1m KClO4) over the range of 4 logarithmic units of
free ZnII. The recorded spectra were processed by nonlinear least-squares
fit analysis using the SPECFIT software package.[38] The resulting binding
affinities are apparent stability constants, and are defined by Equa-
tion (2):
{4-{2-{4-[(Bispyridin-2-ylmethylamino)methyl]-1H-benzimidazol-2-yl}-
phenylsulfamoyl}phenoxy}acetic acid (9b): Ethyl ester 9a (22 mg,
0.033 mmol) was hydrolyzed as described for acid 6b to give free acid 9b
(16 mg, 76%). For all photophysical studies a sample of the compound
(10 mg) was further purified by semipreparative reversed-phase HPLC
(10î300 mm C18 column, CH3CN/H2O (0.01%TFA) 75:25!98:2). M.p.
½ZnLŠ
K0 ¼
ð2Þ
½ZnŠð½LŠ þ ½LHŠ þ ½LH2Š þ ::: ½LHmŠÞ
1
80 828C; H NMR (CDCl3, 400 MHz): d=3.91 (s, 4H), 3.99 (s, 2H), 4.39
in which [ZnL]=concentration of zinc ligand complex, [Zn]=concentra-
tion of unbound ZnII, ([L]+[LH]+ ...[LHm])=concentration of all
ligand species that do not involve ZnII (at given conditions of pH 7.2,
0.1m KClO4, 258C).[50]
(s, 2H), 6.50 (d, J=8.7 Hz, 2H), 7.06 (d, J=7.1 Hz, 1H), 7.16 (t, J=
7.6 Hz, 4H), 7.25 (s, 2H), 7.38 (t, J=7.6 Hz, 1H), 7.52 (d, J=8.7 Hz,
2H), 7.59 (td, J=7.6, 1.4 Hz, 2H), 7.68 (d, J=7.6 Hz, 1H), 7.83(d, J=
8.2 Hz, 1H), 8.00 (d, J=7.6 Hz, 1H), 8.40 ppm (d, J=4.9 Hz, 2H); FAB-
MS (thioglycerol): m/z (%): 635 (100) [M+], 373 (62); FAB-HRMS: m/z
calcd (%) for [M+H]+ C34H31N6O5S: 635.2077; found: 635.2119.
Determination of the apparent ZnII affinity of ligand 8b and 9b: A series
of PIPES-buffered solutions (10mm, pH 7.20, 0.1m KClO4) were pre-
pared that contained HEDTA (10mm) or EGTA (10mm) and between
1 9mm of Zn(OTf)2. The concentration of free ZnII was calculated using
the program Hyss,[51] based on the following published pKa and logK
values for HEDTA, pK1 =9.87, pK2 =5.38, pK3 =2.62, pK4 =1.60,
logK(ZnL)=14.6 (258C, m=0.1), and EGTA, pK1 =9.40, pK2 =8.79,
pK3 =2.70, logK(ZnL)=12.6 (258C, m=0.1).[41] According to Martell and
Smith,[41] the tabulated pKa data must be corrected upward by 0.11 to ac-
count for 0.1m ionic strength. This correction is necessary because the
tabulated pKa values are determined on the basis of the concentration
and not the activity of the hydronium ions. For the calculation of the free
ZnII concentrations, the published pKa values of HEDTA and EGTA
were therefore corrected by 0.11. Ligands 8b or 9b were dissolved in
3.0 mL of the corresponding buffer solutions up to a final concentration
of 10mm. After a 24 h equilibration time, the fluorescence intensity at 400
and 500 nm was determined. The experimental data were analyzed by a
nonlinear least-squares fit algorithm with the following formalisms:
Steady-state absorption and fluorescence spectroscopy: All sample stock
solutions and buffer solutions were filtered through 0.2 mm Nylon mem-
brane filters to remove interfering dust particles or fibers. UV/Vis ab-
sorption spectra were recorded at 258C by using a Varian Cary Bio50
UV/Vis spectrometer with constant-temperature accessory. Steady-state
emission and excitation spectra were recorded with a PTI fluorimeter
and FELIX software. Throughout the titration the sample solution was
stirred with a magnetic stirring device. For all titrations the path length
was 1 cm with a cell volume of 3.0 mL. All fluorescence spectra have
been corrected for the spectral response of the detection system (emis-
sion correction file provided by the instrument manufacturer) and for the
spectral irradiance of the excitation channel (by using a calibrated photo-
diode). Quantum yields were determined by using quinine sulfate dihy-
drate in 1.0n H2SO4 (Ff =0.543ꢁ0.03) as the fluorescence standard.[48]
Electrode calibration in aqueous solution: Measurements were per-
formed with an Orion microcombination glass electrode. The electrode
was calibrated for ꢀlog[H3O+] by titration of a standardized HCl solu-
tion (Aldrich, 0.1n volumetric standard) with KOH (Aldrich, 0.1n volu-
metric standard) at 258C and 0.1m ionic strength (KCl). The endpoint,
electrode potential, and slope were determined by using Gran×s
method[34] as implemented in the software GLEE.[49] The calibration pro-
cedure was repeated three times prior to each pKa value determination.
The electrode potential was measured with the Corning pH/Ion Analyzer
355 and the emf measurements were reproducible with ꢁ0.1 mV accura-
cy.
By assuming a 1:1 metal-to-ligand stoichiometry, the following relation-
ship [Eq. (3)] was found between the free ZnII concentration, apparent
dissociation constant Kd, and fluorescence intensity F:[4]
FꢀFmin
ð3Þ
½ZnŠ ¼ Kd
FmaxꢀF
in which Fmin =fluorescence intensity of free ligand, Fmax =fluorescence
intensity of ZnII-bound ligand.
Using Equation (3) the emission intensity F can be expressed as a func-
tion of free ZnII and Kd, as shown in Equation (4):
Potentiometry: All protonation constants reported in this study were de-
termined from potentiometric titrations and additionally confirmed by
spectrophotomotetric measurements. Potentiometric titrations were car-
ried out with a motorized burette (Dosimat, Metrohm, Switzerland) by
adding a total of 40 100 aliquots of KOH (0.1m) to 10.0 mL of a solution
of the corresponding ligand (0.5 1.0mm) in KCl (0.1m) under a N2 atmos-
phere (solvent-vapor-saturated gas). Throughout the titration, the tem-
perature was maintained at 25ꢁ0.18C by circulating constant-tempera-
ture water through the water-jacket of the titration cell. For the potentio-
metric titration of ligands 7b, 8b, and 9b, the initial solution was further
acidified by adding 1 3molar equivalents of HCl (0.5 m). The emf data
were converted to ꢀlog[H3O+] based on the electrode potential E8 and
slope, which were determined as described above prior to each titration.
The pKa values were obtained from nonlinear least-squares fit analysis of
the potentiometric data.[35]
½ZnŠFmax þ KdFmin
ð4Þ
F ¼
Kd þ ½ZnŠ
Nonlinear least-squares fit analysis of the experimental intensities versus
the (calcd) free ZnII concentrations allowed for the determination of
Fmax, Fmin, and Kd.
Acknowledgements
Financial support from the Georgia Institute of Technology is gratefully
acknowledged. We also thank D. Bostwick for mass spectral data, and J.
Cody for a critical review of the manuscript.
Spectrophotometric titrations: The UV/Vis absorption spectra of the li-
gands were monitored for a series of solutions in which ꢀlog[H3O+] was
3024
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 3015 3025

Zinc(ii)-Selective Sensors
3015 3025
[28] M. Mosquera, J. C. Penedo, M. C. Rios Rodriguez, F. Rodriguez-
Prieto, J. Phys. Chem. 1996, 100, 5398.
[29] S. Santra, G. Krishnamoorthy, S. K. Dogra, J. Phys. Chem. A 2000,
104, 476.
[1] A. P. DeSilva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher, T. E. Rice, Chem. Rev. 1997,
97, 1515.
[2] W. T. Mason, Fluorescent and Luminescent Probes for Biological Ac-
tivity, Academic Press, San Diego, 1999.
[3] K. Rurack, Spectrochim. Acta Part A 2001, 57, 2161.
[4] G. Grynkiewicz, M. Poenie, R. Y. Tsien, J. Biol. Chem. 1985, 260,
3440.
[30] S. Santra, S. K. Dogra, Chem. Phys. 1998, 226, 285.
[31] C. J. Fahrni, M. M. Henary, D. G. VanDerveer, J. Phys. Chem. A
2002, 106, 7655.
[32] M. Bonnat, M. Bradley, J. D. Kolburn, Tetrahedron Lett. 1996, 37,
5409.
[33] W. H. Kwok, H. Zhang, P. Payra, M. Duan, S.-C. Hung, D. H. John-
ston, J. Gallucci, E. Skrzypczak-Jankun, M. K. Chan, Inorg. Chem.
2000, 39, 2367.
[34] G. Gran, Analyst 1951, 77, 661.
[35] A. E. Martell, R. J. Motekaitis, The Determination and Use of Stabil-
ity Constants, VCH, New York, 1992.
[5] R. Y. Tsien, Trends Neurosci. 1988, 11, 419.
[6] R. P. Haugland, Handbook of Fluorescent Probes and Research
Products, Molecular Probes, Eugene, Oregon, 2002.
[7] P. D. Zalewski, S. H. Millard, I. J. Forbes, O. Kapaniris, A. Slavoti-
nek, W. H. Betts, A. D. Ward, S. F. Lincoln, I. Mahadevan, J. Histo-
chem. Cytochem. 1994, 42, 877.
[36] J. K. Romary, J. D. Barger, J. E. Bunds, Inorg. Chem. 1968, 7, 1142.
[37] D. W. Gruenwedel, Inorg. Chem. 1968, 7, 495.
[38] R. A. Binstead, A. D. Zuberb¸hler, SPECFIT Global Analysis
System, Spectrum Software Associates, Marlborough MA 01752,
2001.
[39] V. I. Minkin, A. D. Garnovskii, J. Elguero, A. R. Katritzky, O. V. De-
nisko, Adv. Heterocycl. Chem. 2000, 76, 157.
[40] J. Elguero, C. Marzin, A. R. Katritzky, P. Linda, Adv. Heterocycl.
Chem., Suppl. 1, 1976.
[41] A. E. Martell, R. M. Smith, NIST Critical Stability Constants of
Metal Complexes, NIST Standard Reference Database 46, Ver-
sion 5.0, 1998.
[42] D. A. Deranleau, J. Am. Chem. Soc. 1969, 91, 4044.
[43] J. E. Coleman, K. Nakamura, J. F. Chlebowski, J. Biol. Chem. 1983,
258, 386.
[44] J. E. Coleman, B. L. Vallee, J. Biol. Chem. 1960, 235, 390.
[45] J. A. Hunt, C. A. Fierke, J. Biol. Chem. 1997, 272, 20364.
[46] P. Canonne, M. Belley, G. Fytas, J. Plamondon, Can. J. Chem. 1988,
66, 168.
[47] U. Holzgrabe, Arch. Pharm. 1987, 320, 647.
[48] J. N. Demas, G. A. Crosby, J. Phys. Chem. 1971, 75, 991.
[49] P. Gans, B. O×Sullivan, Talanta 2000, 51, 3 3 .
[50] G. Schwarzenbach, Complexometric Titrations, Interscience, New
York, 1957.
[8] R. D. Palmiter, T. B. Cole, S. D. Findley, EMBO J. 1996, 15, 1784.
[9] R. D. Palmiter, T. B. Cole, C. J. Quaife, S. D. Findley, Proc. Natl.
Acad. Sci. USA 1996, 93, 14934.
[10] T. Budde, A. Minta, J. A. White, A. R. Kay, Neuroscience 1997, 79,
347.
[11] H. J. Wenzel, T. B. Cole, D. E. Born, P. A. Schwartzkroin, R. D. Pal-
miter, Proc. Natl. Acad. Sci. USA 1997, 94, 12676.
[12] M. S. Nasir, C. J. Fahrni, D. A. Suhy, K. J. Kolodsick, C. P. Singer,
T. V. O×Halloran, J. Biol. Inorg. Chem. 1999, 4, 775.
[13] C. W. MacDiarmid, L. A. Gaither, D. Eide, EMBO J. 2000, 19, 2845.
[14] G. Ranaldi, G. Perozzi, A. Truong-Tran, P. Zalewski, C. Murgia,
Am. J. Physiol. 2002, 283, 1365.
[15] H. Haase, D. Beyersmann, Biochem. Biophys. Res. Commun. 2002,
296, 923.
[16] C. P. Kirschke, L. P. Huang, J. Biol. Chem. 2003, 278, 4096.
[17] S. C. Burdette, C. J. Frederickson, W. Bu, S. J. Lippard, J. Am.
Chem. Soc. 2003, 125, 1778.
[18] T. Hirano, K. Kikuchi, Y. Urano, T. Nagano, J. Am. Chem. Soc.
2002, 124, 6555.
[19] K. R. Gee, Z. L. Zhou, W. J. Qian, R. Kennedy, J. Am. Chem. Soc.
2002, 124, 776.
[20] E. Kimura, S. Aoki, E. Kikuta, T. Koike, Proc. Natl. Acad. Sci. USA
2003, 100, 3731.
[21] D. A. Pearce, N. Jotterand, I. S. Carrico, B. Imperiali, J. Am. Chem.
Soc. 2001, 123, 5160.
[51] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca,
Coord. Chem. Rev. 1999, 184, 311.
[22] C. J. Fahrni, T. V. O×Halloran, J. Am. Chem. Soc. 1999, 121, 11448.
[23] K. R. Gee, Z. L. Zhou, D. Ton-That, S. L. Sensi, J. H. Weiss, Cell
Calcium 2002, 31, 245.
[24] S. Maruyama, K. Kikuchi, T. Hirano, Y. Urano, T. Nagano, J. Am.
Chem. Soc. 2002, 124, 10650.
[52] Note added in proof: The following two reports on emission-ratio-
metric zinc-selective fluorescent sensors just appeared in print: M.
Taki, J. L. Wolford, T. V. O×Halloran, J. Am. Chem. Soc. 2004, 126,
712 and C. J. Chang, J. Jaworski, E. M. Nolan, M. Sheng, S. J. Lip-
pard, Proc. Nat. Acad. Sci. 2004, 101, 1129.
[25] M. M. Henary, C. J. Fahrni, J. Phys. Chem. A 2002, 106, 5210.
[26] K. Das, N. Sarkar, D. Majumdar, K. Bhattacharyya, Chem. Phys.
Lett. 1992, 198, 443.
Received: July 3, 2003
Revised: December 24, 2003
[27] H. K. Sinha, S. K. Dogra, Chem. Phys. 1986, 102, 337.
Published online: April 28, 2004
3025
Chem. Eur. J. 2004, 10, 3015 3025
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim