Complexation of Zn2+ by the fluorophore 2-((E)-2-phenyl)ethenyl- 8-(N-4-methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid: A preparative, potentiometric, UV-visible, and fluorescence study

Article abstract of DOI:10.1071/CH10173

The preparation of the Zn²⁺ specific fluorophore 2-((E)-2-phenyl)ethenyl-8-(N-4-methylbenzene-sulfonyl)aminoquinol-6-yloxyacetic acid, H₂3, is described. The protonated form, H₃3 ⁺, is characterized by pK_a<

Full text of DOI:10.1071/CH10173

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2010, 63, 1448–1452
www.publish.csiro.au/journals/ajc
Complexation of Zn2+ by the Fluorophore 2-((E)-2-Phenyl)
ethenyl-8-(N-4-methylbenzenesulfonyl)aminoquinol-
6-yloxyacetic Acid: A Preparative, Potentiometric,
UV-visible, and Fluorescence Study
Hilary C. Coleman,A Bruce L. May,A and Stephen F. LincolnA,B
ASchool of Chemistry and Physics, University of Adelaide, Adelaide, SA 5005, Australia.
BCorresponding author. Email: Stephen.Lincoln@adelaide.edu.au
The preparation of the Zn2+ specific fluorophore 2-((E)-2-phenyl)ethenyl-8-(N-4-methylbenzene-sulfonyl)aminoquinol-
6-yloxyacetic acid, H23, is described. The protonated form, H33+, is characterized by pKa values of 2.71 0.03,
4.92 0.03, and 10.46 0.03 in 25% (v/v) aqueous ethanol 0.10 mol L−1 in NaClO4 at 298.2 K determined by potentio-
metric titration. At pH 6.6, but otherwise under the same conditions, the dianion, 32−, forms the fluorescent complexes
[Zn(3)] and [Zn(3)2]2−, characterized by log(K1/L mol−1) = 10.5 0.20 and log(K2/L mol−1) = 11.1 0.1, respectively,
as determined by fluorimetry. These data are compared with analogous data for the structurally similar and widely used
fluorophore 2-methyl-8-p-toluenesulfonamido-6-quinolyloxyacetic acid (Zinquin A).
Manuscript received: 28 April 2010.
Manuscript accepted: 1 July 2010.
Introduction
Zinc(ii) is the second most abundant transition metal ion in
humans after Fe2+/Fe3+ [1–3]
It is involved in many physiologi-
.
cal processes and is largely found either at the active site or as
a structural component of numerous enzymes.[4–8] Zinc(ii) reg-
ulates RNA and DNA metabolism[9–11] and some neurological
diseases appear to involve Zn2+ metabolic dysfunction.[12–14]
Consequently, the location and quantification of Zn2+ in bio-
logical tissues is of significant importance and has led to the
development of a substantial range of ligands which fluoresce
O2S
NH
NH2
N
N
HO
O
when bound by Zn2+ [14–18]
.
The tissue stain 8-aminoquinoline, 1 (Fig. 1), was the start-
ing point for the development of the widely used Zn(ii) specific
fluorophore, 2-methyl-8-p-toluenesulfonamido-6-quinolyloxy-
aceticacid, H22, anditsethylester(ZinquinAandE)[15,18–26] and
we now report a study of the styryl derivative of H22, 2-((E)-2-
phenyl)ethenyl-8-(N-4-methylbenzenesulfonyl)aminoquinol-6-
yloxyacetic acid, H23, as part of a broad study of the effect of
ligand structural change on the formation of Zn2+ complexes,
and the potential for development of new Zn2+ selective fluoro-
phore designs.
O
H22
1
3a
2a
12
O2S
7
11
13
NH
9
N
10
Results and Discussion
Potentiometric Determination of the pKas of H33+
HO
3
O
5
4
H23
O
2-((E)-2-Phenyl)ethenyl-8-(N-4-methylbenzenesulfonyl)amino-
quinol-6-yloxyacetic acid, H23, was prepared by the hydrol-
ysis of its ethyl ester.[27] Potentiometric titrations of 1.03 ×
10−3 mol L−1 H23 in 25% v/v aqueous ethanol in the presence
of 9.2 × 10−3 mol L−1 HClO4 at I = 0.10 mol L−1 (NaClO4)
with 0.126 mol L−1 NaOH at 298.2 K, show H33+ to
Fig. 1. Structures of 1, H22, and H23.
which are assigned to the sulfonamide, carboxylic, and quino-
linium protons, respectively. These compare with pKa3
=
undergo three acid dissociations characterized by pKa3
10.46 0.03, pKa2 = 4.92 0.03, and pKa1 = 2.71 0.03,
=
10.13 0.08, pKa2 = 4.40 0.02, and pKa1 = 3.08 0.06 for
H32+ under the same conditions.[15b] Thus, the quinolinium
© CSIRO 2010
10.1071/CH10173
0004-9425/10/101448

Complexation of Zn2+
1449
proton of H33+ is more acidic than that of H32+, while the
sulfonamide and carboxylic protons are less acidic. This is con-
sistent with the increased conjugation destabilizing H23+ with
respect to H23 to a greater extent than the destabilization of H32+
with respect to H22. The higher pKa2 and pKa3 characterizing
H23 and H3−, respectively, probably reflect the greater delocal-
ization of the di- and mono-negative charges of their conjugate
bases, H3− and 32−, respectively, and decreased electrostatic
resistance to deprotonation by comparison with H22 and H2−.
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
UV-visible Studies of [Zn(3)] and [Zn(3)2]2− Formation
UV-vis spectra were recorded for 25% v/v aqueous ethanol
solutions buffered at pH 6.6 with 1.0 × 10−3 mol L−1 NaPIPES
buffer at I = 0.10 mol L−1 (NaClO4) and 298.2 K under which
conditions the ligand exists dominantly as H3−. The variation
of the UV-vis spectrum of H3− with increasing [Zn2+
]
is
total
250
300
350
400
450
500
shown in Fig. 2. The best fit of an algorithm for the equilibria
shown in Eqns 1 and 2 to the absorbance variation at 1.0 nm
intervals over the range 270–450 nm yields log(K1/L mol−1) =
11.5 0.76 (K1 = 3.2 × 1011 L mol−1) and log(K2/L2 mol−2) =
9.7 0.84 (K2 = 5.0 × 109 L mol−1). The derived UV-vis spec-
trum of H3− and those of [Zn(3)] and [Zn(3)2]2− obtained
from the fitting procedure are shown in Fig. 3 from which it
is seen that the spectra of [Zn(3)] and [Zn(3)2]2− are shifted
to longer wavelengths by comparison with those of H3− as a
consequence of their greater conjugation. The corresponding
UV-vis spectra of (a) H2− (λmax = 245 nm and ε = 4.1 ×
104 L mol−1 cm−1 andλmax = 336 nmandε = 4.2 × 103 L mol−1
cm−1); (b) [Zn(2)] (λmax = 264 nm and ε = 3.6 × 104 L mol−1
cm−1 and λmax = 361 nm and ε = 3.9 × 103 L mol−1 cm−1); and
(c) [Zn(2)2]2− (λmax = 264 nm and ε = 9.3 × 104 L mol−1 cm−1
and λmax = 361 nm and ε = 7.8 × 104 L mol−1 cm−1) are con-
sistent with their lesser conjugation by comparison with H3−,
Wavelength [nm]
Fig. 2. Variation of the UV-vis spectrum of 1.82 × 10−5 mol L−1 H3− in
25% v/v aqueous ethanol buffered 0.10 mol L−1 in NaClO4 buffered at
pH 6.6 (1.0 × 10−3 mol L−1 NaPIPES) at 298.2 K with [Zn2+
]
−i1n the
total
range 3.9 × 10−7 mol L−1 (= [Zn2+
]
adventitious) to 1.72 × 10−4 mol L .The
arrows show the direction of absorbance change with increase in [Zn2+
Isosbestic points occur at 305, 336, and 375 nm.
]
.
total
6
5
4
3
2
1
0
(c)
(b)
[Zn(3)], and [Zn(3)2]2−, respectively, resulting in shorter λmax
.
(a)
H3−1 + Zn+2 ↔ H+ + [Zn(3)]
(1)
(2)
H3− + [Zn(3)] ↔ H+ + [Zn(3)2]2−
Fluorimetric Studies of [Zn(3)] and [Zn(3)2]2− Formation
250
300
350
400
450
500
Under the same conditions as those of the UV-vis stud-
Wavelength [nm]
ies the fluorescence of H3− increases with increase in
[Zn2+
]
as shown in Fig. 4. The best fit of an algo-
Fig. 3. The UV-vis spectra of (a) H3− (λmax = 303 nm and ε = 3.8 ×
104 L mol−1 cm−1 and λmax = 366 nm and ε = 1.9 × 104 L mol−1 cm−1);
total
rithm for the sequential equilibria represented by Eqns 1
(b) [Zn(3)] (λmax = 313 nm and ε = 4.8 × 104 L mol−1 cm−1 and λmax
=
and
2 to the fluorescence variation at 0.5 nm inter-
378 nm and ε = 1.5 × 104 L mol−1 cm−1); and (c) [Zn(3)2]2− (λmax
=
vals over the range 430–650 nm yields log(K1/L mol−1) =
10.5 0.20 (K1 = 3.2 × 1010 L mol−1) and log(K2/L mol−1) =
11.1 0.1 (K2 = 1.3 × 1011 L mol−1). Because of the greater
312 nm and ε = 5.2 × 104 L mol−1 cm−1 and λmax = 376 nm and ε = 1.8 ×
104 L mol−1 cm−1) derived from the fitting of an algorithm for the molar
absorbance variation with [Zn2+
in the text.
]total and equilibria (1) and (2) as described
changes in the fluorescence spectra as [Zn2+
]
increases
total
by comparison with the changes and in the UV-vis spec-
tra and the greater wavelength range of 220 nm suitable for
K1 and K2 derivation from the fluorescence data compared
with the 180 nm wavelength range for the UV-vis data, the
K1 and K2 obtained by fluorimetry are considered the more
accurate. Their similar magnitudes are consistent with the coor-
dination number of [Zn(3)] changing from six (where four
aqua ligands share the first coordination sphere and 32− is
coordinated as a bidentate ligand through the sulfonamide
and quinol nitrogens) to four in tetrahedral [Zn(3)2]2− as a
consequence of steric interactions between the two 32− lig-
ands and the coordinative and stereochemical flexibility of d10
log(K2/L mol−1) = 8.8, which is consistent with Zn2+ retaining
six-coordination in both complexes because of the smaller size
of 22− [15b]
.
The fluorescence intensity of the spectrum of [Zn(3)] derived
from the fitting procedure applied to the data in Fig. 4 is slightly
greater than that of [Zn(3)2]2− (Fig. 5), which is consistent with
a similar relationship for [Zn(2)] and [Zn(2)2]2− [15b] and the
broader observation that the fluorescence of 22− in complexes of
the general formula [Zn(2)L] varies significantly with the nature
of L.[18] The fluorescence quantum yield, φ, of 32− in [Zn3]
is 0.14. The corresponding fluorescence spectra of (a) [Zn(2)]
Zn2+. For [Zn(2)], log(K1/L mol−1) = 10.5 and for [Zn(2)2]2−
,

1450
H. C. Coleman, B. L. May, and S. F. Lincoln
700
600
500
400
300
200
100
0
O2S
NH
N
O
O
O2S
NH
N
430
480
530
580
630
Wavelength [nm]
O
O
Fig. 4. Increase in fluorescence of 5.56 × 10−6 mol L−1 H3− in 25%
v/v aqueous ethanol 0.10 mol L−1 in NaClO4 buffered at pH 6.6
Fig. 6. Photoisomerization of E-H3− to Z-H3−.
(1.0 × 10−3 mol L−1 NaPIPES) at 298.2 K with [Zn2+
]
in the range
total
3.9 × 10−7 mol L−1 (= [Zn2+
]
adventitious) to 5.0 × 10−5 mol L−1. Excitation
at 336 nm with excitation and emission slit widths of 5 mm.
those of [Zn(3)] and [Zn(3)]2− (Fig. 5). Under the conditions of
the equilibrium studies [Zn2+
]
adventitious = 3.9 × 10−7 mol L−1
,
14
12
(a)
which was added to the experimentally added [Zn2+] to give
[Zn2+
]
used in the K1 and K2 fluorimetric and UV-vis
total
(b)
determinations.
The absorbance spectrum of H3− in the absence of adventi-
tious Zn2+ is shown in Fig. 3. Under the same conditions H3−
showsnofluorescence.Thefluorescenceof 32− inducedthrough
coordination in [Zn(3)] and [Zn(3)]2− occurs through a chela-
tion enhanced fluorescence (CHEF) mechanism,[28] whereby
the coordination of the amide and quinoline nitrogens to Zn2+
forms a structurally stiffening five-membered chelate ring,
which diminishes fluorescence quenching by energy dissipation
throughvibrationalandrotationalmodes.Thenitrogencoordina-
tion also decreases any residual photoinduced electron transfer
effects.[29,30]
10
8
6
4
2
0
430
480
530
580
630
AqualitativeUV-visstudyshowsthat3− bindstoCo2+, Ni2+
,
Wavelength [nm]
Cu2+, and Cd2+ but not to Ca2+, however, the complexes of the
first three metals are not fluorescent probably due to quenching
through electronic transitions occurring in their d7, d8, and d9
manifolds. In contrast, the d10 Cd2+ complex, in which such
quenching cannot occur, is fluorescent. However, in healthy cells
the concentration of Cd2+ is very low by comparison with that
of Zn2+ and so is unlikely to interfere significantly in the use of
Zn2+ fluorophores in cellular and tissue studies.[31]
Fig. 5. The fluorescence spectra of (a) [Zn(3)] (λmax = 534 nm and
molar fluorescence = 1.26 × 108 L mol−1) and (b) [Zn(3)]2− per 32− lig-
and (λmax = 537 nm and molar fluorescence = 1.18 × 108 L mol−1) derived
from the fitting of an algorithm for the fluorescence variation with [Zn2+
and equilibria (1) and (2) as described in the text.
]
total
(λmax = 483 nm and molar fluorescence = 5.56 × 107 L mol−1
)
and (b) [Zn(2)]2− per 32− ligand (λmax = 483 nm and molar
Photoisomerism of H3−
fluorescence = 6.17 × 107 L mol−1) reflect their lesser conjuga-
[15a]
tion through their shorter λmax
.
A change in the solution spectrum of E-H3− occurs upon con-
tinuous exposure to laboratory light and is attributed to photo-
isomerization about the ethenyl bond to form Z-H3− (Fig. 6)
as shown in Fig. 7. The small absorbance decrease is complete
after 15 min when a photostationary state is reached in which
the proportions of the E-H3− and Z-H3− isomers are unknown.
This change reverses after refluxing in darkness with a catalytic
amount of p-toluenesulfonic acid. To avoid any contribution to
the above discussed UV-vis and fluorimetric equilibrium studies,
all solution manipulation involving E-H3− was carried out under
reduced light, and all solutions were contained in foil-wrapped
vessels and allowed to equilibrate for at least 30 min at 298.2 K
in darkness before spectroscopic measurement.
At the low [H3−]total and [Zn2+
]
total of the fluorimetric study
adventitious, aris-
the low levels of impurity, or adventitious, [Zn2+
]
ing dominantly from the NaClO4 supporting electrolyte and the
NaPIPES buffer become significant by comparison with the
added [Zn2+] and were included in the [Zn2+
]
total in the UV-vis
and fluorimetric equilibrium studies.[15,19] The [Zn2+
]
adventitious
was determined in the 25% v/v aqueous ethanol 0.10 mol L−1
in NaClO4 buffered at pH 6.6 (1.0 × 10−3 mol L−1 NaPIPES)
in which all solutions for UV-vis and fluorometric studies were
made. This determination was made using an EDTA titration
method described in the experimental section, which shows the
fluorescence of H3− to be negligibly low by comparison with

Complexation of Zn2+
1451
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Other Materials
Sodium
piperazine-N,Nꢀ-bis[2-ethanesulfonate]
buffer,
NaPIPES (Cal Biochem), NaOH (Ajax) and HClO4 (70% in
water, Ajax), and EDTA disodium salt (Na2EDTA·H2) (Ajax),
were used as received. Metal perchlorate salts were either pur-
chased (Fluka) or prepared by reaction of the corresponding
metal carbonate with a stoichiometric amount of perchloric
acid. All perchlorates were recrystallized from ethanol/water,
vacuum-dried, and stored over P2O5. Aqueous metal perchlo-
rate solutions were standardized in triplicate by passing down a
DowexAg 50W-X2 cation exchange resin (acid form) 2 × 20 cm
columnandback-titratingtheacidinthecollectedeluentsolution
with aqueous NaOH solution. The NaOH solution was standard-
ized against potassium phthalate. Quinine hemisulfate (Aldrich)
was dried under vacuum at room temperature for 12 h and then
stored over P2O5 before use in quantum yield determinations.
Analytical grade ethanol was purified by fractional distillation
under nitrogen. Deionized water was ultrapurified with a Milli-
Q Reagent system to produce water with a specific resistance of
>15 Mꢀ cm and then boiled to remove CO2.
250
300
350
400
450
500
Wavelength [nm]
Fig. 7. The absorbance time dependence during exposure to daylight of
E-H3− in 25% v/v aqueous ethanol 0.10 mol L−1 in NaClO4 buffered at pH
6.6 (1.0 × 10−3 mol L−1 NaPIPES) at 298.2 K.
Glassware and instruments used in transferring, storing,
preparing, and measuring the properties of the final product
(including pipettes, cuvettes, and potentiometric titration cells)
were thoroughly washed in Decon 90, rinsed several times,
and then soaked overnight in Milli-Q water and oven-dried
before use. Micropipettes (HTL) were used for all volume
measurements under 2 mL.
Conclusion
The K1 value for [Zn(3)] is similar to that of [Zn(2)] while the K2
valuefor[Zn(3)]2− is200timeslargerthanthatof[Zn(2)]2− [15b]
.
This is consistent with the greater conjugation of 32− causing
it to be a more effective bidentate Lewis base for Zn2+ than
is 22−. However, H23 is not an ideal fluorophore for routine
analytical use due to the photoisomerization of E-3 to Z-3 in
solution in daylight unless care is taken to avoid it. Nevertheless,
this study demonstrates the effect of increased conjugation in the
quinoline-based fluorophores and provides insight for further
Zn2+ selective fluorophore development.
Potentiometric Titrations
Potentiometric titrations were performed using a Metrohm 800
Dosino equipped with a 5 mL burette and an Orion Ross semi-
micro combination pH electrode, which contained 0.10 mol L−1
NaClO4 in 25% v/v aqueous ethanol. Data collection was
controlled by the program Tiamo running off an IBM per-
sonal computer interfaced to the pH electrode through a 809
Titrando potentiometer. All titrations were thermostatted at
298.2 0.1 K in a foil wrapped water-jacketed titration vessel
that was closed to the atmosphere. Nitrogen was first bubbled
through 0.10 mol L−1 KOH to remove CO2 and then through
0.10 mol L−1 NaClO4 to remove any KOH spray and to saturate
it with solvent (25% v/v aqueous ethanol) before it was passed
through the magnetically stirred titration solution before and
during the titration.
Experimental
Materials
Preparation of 2-((E)-2-Phenyl)ethenyl-8-
(N-4-methylbenzenesulfonyl)aminoquinol-
6-yloxyacetic Acid, H23
Asolutionofethyl-2-(2-[(E)-2-phenyl-1-ethenyl]-6-quinolyl-
oxy-8-p-toluenesulfonamido)acetate[27] (300 mg, 0.586 mmol)
and sodium hydroxide (292 mg, 7.3 mmol) in ethanol (20 mL)
was heated at reflux for 18 h. The mixture was cooled to room
temperature and diluted with 10% w/v aqueous citric acid solu-
tion (20 mL). The precipitated solid was collected by vacuum
filtration and washed with water (2 × 10 mL). The product crys-
tallized from aqueous ethanol as pale orange needles (174 mg,
61%), mp 230◦C (dec). νmax (nujol)/cm−1 3176 (b), 2582 (b),
1739 (s), 1625, 1600, 1596, 1504, 1209, 1162 (s), 1097, 960,
900, 858, 836, 690, 665. δH (d6-DMSO) 9.9 (br s, 1H, NH), 8.15
(d, J 10.0, 1H, quinolyl H4), 7.87 (d, J 15.8, 1H, ethenyl H9),
7.84 (d, J 8.0, 2H, tosyl H2a, H2aꢀ), 7.81 (d, J 10, 1H, quinolyl
H3), 7.73 (d, J 7.6, 2H, styryl H11, H11ꢀ), 7.45 (d, J 15.8, 1H,
ethenyl H10), 7.44 (d, J 7.6, 2H, styryl H12, H12ꢀ), 7.36 (m, 1H,
styryl H13), 7.29 (d, J 8, 2H, tosyl H3a, H3aꢀ), 7.26 (d, J 2.8, 1H,
quinolyl H7), 6.77 (d, J 2.8, 1H, quinolyl H5), 4.75 (s, 2H, CH2),
2.25 (s, 3H, CH3). δC (d6-DMSO) 169.7, 155.3, 152.4, 143.7,
136.5, 136.4, 135.7, 134.7, 133.8, 129.7, 128.9, 128.6, 128.3,
127.8, 127.1, 126.9, 121.1, 108.8, 101.9, 64.8, 20.9. Anal. Calc.
for C26H22N2O5S: C 65.81, H 4.67, N 5.90. Found C 65.88,
H 4.71, N 5.89%.
All solutions were prepared in 25% v/v aqueous ethanol and
were 0.10 mol L−1 in NaClO4. Potentiometric titrations were
performed by the titration of 2.0 mL aliquots of a solution con-
taining ethyl-2-(2-[(E)-2-phenyl-1-ethenyl]-6-quinolyloxy-8-p-
toluenesulfonamido)acetic acid, H23 (1.03 × 10−3 mol L−1),
HClO4 (9.21 × 10−3 mol L−1), and NaClO4 (I = 0.1 mol L−1
)
with 0.126 mol L−1 NaOH (standardized by titration of
2 mL aliquots with 0.010 mol L−1 potassium phthalate). Cal-
ibrations were performed daily by titrating 2.0 mL of
a 9.21 × 10−3 mol L−1 HClO4 solution with standardized
0.126 mol L−1 NaOH, where both solutions were 0.10 mol L−1
in NaClO4. An algorithm for the pH variation of a tribasic acid
with added NaOH was fitted to the potentiometric data to derive
the three pKas using the Tiamo software.
Spectroscopic Measurements
All solutions for spectroscopic study were prepared in
25% v/v aqueous ethanol, 0.10 mol L−1 in NaClO4 and
1.00 × 10−3 mol L−1 in NaPIPES buffer at pH 6.6. UV-vis
spectra were recorded with aVarian Cary 300 spectrophotometer

1452
H. C. Coleman, B. L. May, and S. F. Lincoln
using matched quartz cells with a path length of 1 cm in a cell
block thermostatted at 298.2 0.1 K. Samples were equilibrated
for 10 min at this temperature before measurement. All spectra
were obtained over a range of 250–450 nm with a slit width of
2 nm, a scan rate of 600 nm min−1 and a data collection interval
of 1.0 nm.
Fluorescence spectra were recorded on aVarian Cary Eclipse
fluorimeter. The solutions were contained in a 1 cm pathlength
quartz cell and placed in a thermostatted 298.2 K sample block
for 10 min before measurement. Spectra were obtained over a
range of 430–650 nm with excitation at the isosbestic point
at 336 nm (Figs 2 and 3). This isosbestic point was chosen
as the excitation wavelength to ensure that any fluorescence
changes were not due to differing absorbance characteristics
of H3−, [Zn3], and [Zn32]2−. The excitation and emission slit
widths were 5 nm, the scan rate was 120 nm min−1 and the data
collection interval was 1.0 nm. The quantum yields[32] were
determined using quinine hemisulfate as the reference fluoro-
phore and the refractive index of the solvent system was obtained
from literature data.[33]
[6] D. H. Nies, Science 2007, 317, 1695. doi:10.1126/SCIENCE.1149048
[7] E. L. Que, D. W. Domaille, C. J. Chang, Chem. Rev. 2008, 108, 1517.
doi:10.1021/CR078203U
[8] W. Maret,Y. Li, Chem. Rev. 2009, 109, 4682. doi:10.1021/CR800556U
[9] A. Nomura, Y. Sugiura, J. Am. Chem. Soc. 2004, 126, 15374.
doi:10.1021/JA045663L
[10] M. Nagaoka,Y. Doi, J. Kuwahara,Y. Sugiura, J.Am. Chem. Soc. 2002,
124, 6526. doi:10.1021/JA025856D
[11] C. I. Stains, J. R. Porter, A. T. Ooi, D. J. Segal, I. Ghosh, J. Am. Chem.
Soc. 2005, 127, 10782. doi:10.1021/JA051969W
[12] M. P. Cuajungco, G. J. Lees, Neurobiol. Dis. 1997, 4, 137.
doi:10.1006/NBDI.1997.0163
[13] L. E. Scott, C. Orvig, Chem. Rev. 2009, 109, 4885. doi:10.1021/
CR9000176
[14] M. Nolan, S. J. Lippard, Acc. Chem. Res. 2009, 42, 193. doi:10.1021/
AR8001409
[15] (a) K. M. Hendrickson, T. Rodopoulos, P.-A. Pittet, I. Mahadevan,
S. F. Lincoln, A. D. Ward, T. Kurucsev, P. A. Duckworth, I. J. Forbes,
P. D. Zalewski, W. H. Betts, J. Chem. Soc., Dalton Trans. 1997, 3879.
doi:10.1039/A703433D
(b) K. Hendrickson, PhD Thesis: The use and spectroscopic proper-
ties of Zinquin, a zinc(II) specific fluorophore 1999 (University of
Adelaide: Adelaide).
The K1 and K2 values were derived by fitting algorithms
describing the variation of absorbance and fluorescence with
equilibria (1) and (2) at 1 nm intervals over the ranges 270–
450 nm and 430–650 nm, respectively, using the SPECFIT/32
protocol.[34]
[16] S. C. Burdette, C. J. Frederickson, W. Bu, S. J. Lippard, J. Am. Chem.
Soc. 2003, 125, 1778. doi:10.1021/JA0287377
[17] T. Hirano, K. Kikuchi, Y. Urano, T. Nagano, J. Am. Chem. Soc. 2002,
124, 6555. doi:10.1021/JA025567P
[18] K. M. Hendrickson, J. P. Geue, O. Wyness, S. F. Lincoln, A. D. Ward,
J. Am. Chem. Soc. 2003, 125, 3889. doi:10.1021/JA020685Y
[19] S. G. Schulman, L. B. Sanders, Anal. Chim. Acta 1971, 56, 83.
doi:10.1016/S0003-2670(01)80111-2
[20] P. D. Zalewski, I. J. Forbes, W. H. Betts, Biochem. J. 1993, 296, 403.
[21] P. D. Zalewski, I. J. Forbes, R. F. Seamark, R. Borlinghaus, W. H. Betts,
S. F. Lincoln,A. D.Ward, Chem. Biol. 1994, 1, 153. doi:10.1016/1074-
5521(94)90005-1
[22] P. Coyle, P. D. Zalewski, J. C. Philcox, I. J. Forbes, A. D. Ward,
S. F. Lincoln, I. Mahadevan, A. M. Rofe, Biochem. J. 1994, 303, 781.
[23] P. D. Zalewski, S. H. Millard, I. J. Forbes, O. Kapaniris, A. Slavotinek,
W. H. Betts, A. D. Ward, S. F. Lincoln, I. Mahadevan, J. Histochem.
Cytochem. 1994, 42, 877.
[24] P. D. Zalewski, X. Jian, L. L. L. Soon, W. G. Breed, R. F. Seamark,
S. F. Lincoln, A. D. Ward, F.-Z. Sun, Reprod. Fertil. Dev. 1996, 8,
1097. doi:10.1071/RD9961097
[25] I. B. Mahadevan, M. C. Kimber, S. F. Lincoln, E. R. T. Tiekink,
A. D. Ward, W. H. Betts, I. J. Forbes, P. D. Zalewski, Aust. J. Chem.
1996, 49, 561.
[26] P. Zalewski, A. Truong-Tran, S. F. Lincoln, A. D. Ward, A. Shankar,
P. Coyle, L. Jayaram, A. Copley, D. Grosser, C. Murgia, C. Lang,
R. Ruffin, Biotechniques 2006, 40, 509. doi:10.2144/06404RR02
[27] M. C. Kimber, I. B. Mahadevan, S. F. Lincoln, E. R. T. Tiekink, J. Org.
Chem. 2000, 65, 8204. doi:10.1021/JO000678X
The concentration of adventitious Zn2+ in 25% v/v
aqueous/ethanol solutions 0.10 mol L−1 in NaClO4 and
1.00 × 10−3 mol L−1 in NaPIPES buffer at pH 6.6 was deter-
mined by back titration with Na2EDTA·H2 for which a Zn2+
complexation constant K1 = 1016.44 L mol−1 in aqueous solution
is reported.[35] A series of solutions 5.56 × 10−6 mol L−1 in H3−
was prepared in which [Na2EDTA·H2] was gradually increased,
and their fluorescence was determined over the range 430–
650 nm with excitation at 336 nm. Fluorescence decreased with
an increase in [EDTA] and extrapolation to zero fluorescence
intensity yielded a value of 3.9 × 10−7 mol L−1, which was also
taken as the value of [Zn2+
]
assuming stoichiometric
.
adventitious
1:1 complexation of Zn2+ by EDTA4−
Accessory Publication
Fig. A1 shows the potentiometric titration curve for H33+ and
the best-fit curve; Fig. A2 shows the speciation of H33+, H23,
H3−, and 32− with pH; Fig.A3 shows the increase in absorbance
as [Zn2+
increase in fluorescence as [Zn2+
]
total increases and the best fit curve; Fig. A4 shows the
increases and the best fit
]
total
curve; and Fig. A5 shows the speciation of H23, [Zn(3)], and
[Zn(3)2]2− derived from the fluorescence data. All figures are
available on the Journal’s website.
[28] A. W. Czarnik, Acc. Chem. Res. 1994, 27, 302. doi:10.1021/
AR00046A003
[29] D. G. Whitten, Acc. Chem. Res. 1980, 13, 83. doi:10.1021/
AR50147A004
[30] M. R. Wasielewski, Chem. Rev. 1992, 92, 435. doi:10.1021/
CR00011A005
[31] M. Schmid, S. Zimmermann, H. F. Krug, B. Sures, Environ. Int. 2007,
33, 385. doi:10.1016/J.ENVINT.2006.12.003
Acknowledgements
The award of an Australian Postgraduate award to H. Coleman and funding
of this research by the University ofAdelaide is gratefully acknowledged.We
thank Dr D.-T. Pham, Ms. C. Baker, and Dr J. Cawthray for their helpful input.
[32] W. H. Melhuish, J. Phys. Chem. 1961, 65, 229. doi:10.1021/
J100820A009
[33] Handbook of Chemistry and Physics (Eds R. C. Weast, S. M. Selby)
1977 (CRC Press: Cleveland, OH).
[34] R. A. Binstead, B. Jung, A. D. Zuberbuhler, SPECFIT/32, v3.0.39(b)
2007 (Spectrum Software Associates: Marlborough, MA).
[35] R. M. Smith,A. E. Martell, Critical Stability Constants,Vol. 2:Amines
1975 (Plenum Press: New York, NY).
References
[1] C. F. Mills, Zinc in Human Biology 1989 (Springer-Verlag: London).
[2] J. R. R. Fausto da Silva, R. J. P. Williams, The Biological Chemistry
of the Elements 1992 (Oxford University Press: Oxford).
[3] B. L. Vallee, D. S. Auld, Acc. Chem. Res. 1993, 26, 543. doi:10.1021/
AR00034A005
[4] E. Kimura, Acc. Chem. Res. 2001, 34, 171. doi:10.1021/AR000001W
[5] C. Andreini, L. Banci, I. Bertini, A. Rosato, J. Proteome Res. 2006, 5,
3173. doi:10.1021/PR0603699