Selective signalling of zinc ions by modulation of terbium luminescence

Article abstract of DOI:10.1039/b000283f

Luminescence enhancement of terbium emission accompanies zinc ion binding at pH 7.4 with an apparent dissociation constant of 0.6 μM in a competitive ionic background.

Full text of DOI:10.1039/b000283f

Selective signalling of zinc ions by modulation of terbium luminescence†
Ofer Reany,a Thorfinnur Gunnlaugssonab and David Parker*a
a Department of Chemistry, University of Durham, South Road, Durham, UK DH1 3LE.
E-mail: david.parker@durham.ac.uk
b Department of Chemistry, Trinity College Dublin, Dublin 2, Eire
Received (in Cambridge, UK) 10th January 2000, Accepted 16th February 2000,
Published on the Web, 2nd March 2000
Luminescence enhancement of terbium emission accom-
panies zinc ion binding at pH 7.4 with an apparent
dissociation constant of 0.6 mM in a competitive ionic
background.
nide complexes, [LnL2].12 Measurements of the rate constants
for decay of the Eu or Tb excited state were made in H2O and
D2O in the absence and presence of 10 mM ZnCl2. Values for
[EuL2] (kH O = 1.61, kD O = 0.45 ms21; q (Ln hydration state)
2
2
= 1.013) were unchanged in the presence of zinc and data for
[TbL2] (kH O = 0.55, kD O = 0.29; q = 1.0) were similarly
unaffected 2by addition of2ZnCl2 or CaCl2.
Responsive luminescent lanthanide complexes have been
defined recently in which the concentration variation of certain
analytes has been signalled by changes in emission intensity,1
lifetime1,2 or polarisation.3 For this purpose, kinetically robust
complexes of Eu and Tb have been studied in particular and
systems that respond to changes in pH,4 pO22,5 and pX6 in water
have been reported. We set out to prepare analogous complexes
which respond sensitively and selectively to changes in metal
ion concentration, in competitive aqueous media at physio-
logical pH. The signalling of zinc ion concentration is of
particular interest in this respect. Although total [Zn2+] have
long been established to be ca. 12 mM in serum,7 there is
considerable interest in defining the concentration of ‘available’
Zn2+—both in the extracellular and intracellular environments.
Different approaches have been adopted,8,9 of which the most
advanced involve the use of fluorescent 8-tosylamide-quinoline
derivatives.10,11 In seeking to prepare a suitable complex, the
requirement for selectivity over Na+, K+ and more particularly
Mg2+ (ca. 0.9 mM) and Ca2+ (1.2 mM extracellular; < 0.1 mM
intracellular) was evident. Accordingly, we resolved to prepare
the ligand L1a and explore the complexation behaviour of the Eu
and Tb complexes in the macrocyclic derivative L2, as model
systems for selective zinc binding.
Addition of ZnCl2 to L1a or [LnL2] was monitored by changes
in absorption, fluorescence and lanthanide emission at pH 7.4.
The ligand L1a absorbed at 248 nm and no change to the position
and intensity of this band occurred on ion binding, suggesting
that the aryl ether oxygen was not participating in ion binding.
For [TbL2], a small blue-shift was observed in absorption (lmax
255 ? 250 nm); in fluorescence emission two bands were
observed at 440 nm and a second, half as intense at 365 nm,
which may be ascribed to internal charge transfer and locally
excited states, respectively. On binding zinc, the former band
shifted (442 ? 430 nm) and reduced in intensity, while the
latter did not shift but increased in intensity. Zinc binding was
also characterised by a small increase in absorbance (lmax.
=
250 nm) and with a larger enhancement of lanthanide emission
intensity, following excitation at the isosbestic wavelength
(262 nm). Smaller changes in absorption and emission spectra
were obtained following addition of CaCl2 (e.g. < 3 nm shift in
absorption lmax.) and no significant variations in absorption
wavelength were observed following MgCl2 addition, con-
sistent with reduced or insignificant aryl ether oxygen donation,
in these cases. The enhancement of Tb (and Eu) emission
intensity (26 and 42%, respectively) upon Zn2+ binding is likely
to be related to suppression of a photo-induced electron transfer
from the benzylic nitrogen to the intermediate aryl singlet
excited state.1,2 In addition for [EuL2], the metal based emission
and ligand based fluorescence intensity was ca. 20 times lower
than for [TbL2], owing to quenching of the intermediate singlet
state by the Eu3+ ion.
Such spectral changes allowed titrations to be carried out,
monitoring absorption and luminescence intensity variations as
a function of added metal salt concentration. The data obtained
reveal a consistent selectivity pattern (Table 1), with Zn2+
bound more strongly than Ca2+ or Mg2+ ions. Two other general
features emerged from this analysis: metal ion complexation of
the linking amide carbonyl enhanced the ion-binding affinity of
the potentially pentadentate ligand and a slightly higher
apparent affinity was also found in the excited state for the
lanthanide complexes, i.e. when observing ligand or lanthanide
emission intensity variations. The lanthanide ion may serve to
promote intramolecular charge transfer involving conjugation
of the ether oxygen lone pair in the ground and excited states,
via a through-ring electron withdrawing effect. This may also
lead to a better orientation of the oxygen lone pair, in those cases
(e.g. Zn2+ binding) where chelation of the benzylic N and the
aryl ether O occurs. Such behaviour allowed zinc concentra-
tions to be monitored in the sub-micromolar range, even in a
simulated extracellular environment, i.e. the presence of
0.9 mM MgCl2, 1.26 mM CaCl2, 140 mM NaCl and 4 mM KCl,
with an apparent dissociation constant of 0.6 mmol (pH 7.4,
295 K). The observed zinc ion sensitivity and selectivity with
[TbL2] augur well for the development of practicable signalling
Stepwise alkylation of o-aminomethylphenol (BrCH2CO2Et,
5% KI, Na2HPO4; O-alkylation with BrCH2CO2Et, KI, K2CO3)
followed by mild nitration (HNO3–HOAc; 220 °C) yielded an
intermediate nitro-ester which was reduced (H2/Pd–C/MeOH)
and acylated with chloroacetyl chloride (Et3N, DEE) to yield
the triethyl ester L1b. Basic hydrolysis of L1b (pH 12, 20 °C, aq.
NaOH) afforded the amino acid L1a which was purified by ion-
exchange chromatography. Reaction of L1b with 1,4,7-tris(tert-
butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane
(MeCN, K2CO3) followed by treatment with CF3CO2H–
CH2Cl2 and complexation with Ln(NO3)3·5H2O (Ln = Eu, Tb;
pH 5.5; 20 °C) gave the neutral lanthanide complexes;
successive treatment with aqueous sodium hydroxide and then
strong-acid cation exchange resin afforded the desired lantha-
† Electronic supplementary information (ESI) available: representative
examples of spectral changes and of data analysis. See http://www.rsc.org/
suppdata/cc/b0/b000283f/
DOI: 10.1039/b000283f
Chem. Commun., 2000, 473–474
This journal is © The Royal Society of Chemistry 2000
473

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Table 1 Affinity constants (log bML) for cation binding, based on
modulationc of absorption (S0) or lanthanide (Ln*) luminescenced (295 K,
10 mM HEPES, 20 mM NaCl, 115 mM KCl, pH 7.4a)
perturbing Ln luminescence in methanol: A. P. de Silva, H. Q. N.
Gunaratne, T. E. Rice and S. Stewart, Chem. Commun., 1997, 1891.
2 D. Parker, K. Senanayake and J. A. G. Williams, J. Chem. Soc., Perkin
Trans. 2, 1998, 2129.
3 R. S. Dickins, T. Gunnlaugsson, D. Parker and R. D. Peacock, Chem.
Commun., 1998, 1643.
4 T. Gunnlaugsson and D. Parker, Chem. Commun., 1998, 511; T.
Gunnlaugsson, D. Parker and D. A. McDonaill, Chem. Commun., 2000,
93.
5 D. Parker and J. A. G. Williams, Chem. Commun., 1998, 245.
6 D. Parker, K. Senanayake and J. A. G. Williams, Chem. Commun.,
1997, 1777.
Ligand/complex (parameter)
Zn2+
Ca2+
Mg2+
L1(S0)
5.04
5.48
6.35b
5.38
5.99
3.91
3.84
4.03
3.90
4.06
2.1
2.0
2.5
2.1
1.9
[TbL2](S0)
[TbL2](Tb*)
[EuL2](S0)
[EuL2](Eu*)
a Potentiometric titration of L1 (298 K, I = 0.1 M NMe4NO3) revealed a
pKa for N-protonation of 7.35, echoed by the pH variation of ligand
absorption; for binding constant measurements, the pH was adjusted to 7.4
by addition of ‘Tris’ base and was also monitored at the end of the titration.
b In a simulated extracellular environment (Na, K, Mg, Ca), the apparent
binding affinity for [TbL2], based on Tb emission intensity changes, was
6.22. c Binding constants were obtained by iterative least-squares fitting to
a 1:1 model and showed reasonably good agreement to Hill plot analysis.
d Excitation of the Ln complexes was effected at the isosbestic wavelength
(lexc = 262 nm), while absorption spectral changes were monitored at 240
or 250 nm (294 nm for L1). Excitation was also performed in the tail at 320
nm and emission variation as a function of [M2+] gave slightly higher
binding affinities, e.g. for Ca2+; 4.37 with [TbL2] and 4.67 with [EuL2] with
a luminescence enhancement of 20%.
7 M. M. Parker, F. L. Humaller and D. J. Mahler, Clin. Chem., 1967, 13,
40.
8 J. D. Stewart, V. A. Roberts, M. W. Crowder, E. D. Getzoff and S. J.
Benkovic, J. Am. Chem. Soc., 1994, 116, 415; A. Godwin and J. M.
Berg, J. Am. Chem. Soc., 1996, 118, 6514; P. S. Eis and J. R. Lakowicz,
Biochemistry, 1993, 32, 7981; B. Imperiali and G. K. Walkup, J. Am.
Chem. Soc., 1997, 119, 3443.
9 F. Barigeletti, L. Flamigni, G. Calogero, L. Hammerström, J.-P.
Sauvage and J.-P. Collin, Chem. Commun., 1998, 2333.
10 P. D. Zalewski, I. J. Forbes and W. H. Betts, Biochem. J., 1993, 296,
403; P. Coyle, P. D. Zalewski, J. C. Philcox, I. J. Forbes, A. D. Ward,
S. F. Lincoln, I. Mahadevan and A. M. Rofe, Biochem. J., 1994, 303,
781.
11 K. M. Hendrickson, T. Rodopoulos, P.-A. Pilfet, I. Mahadevan, S. F.
Lincoln, A. D. Word, T. Kurucsev, P. A. Duckworth, I. J. Forbes, P. D.
Zalewski and W. H. Betts, J. Chem. Soc., Dalton Trans., 1997, 3879.
12 Complexes gave accurate masses (ESMS2), 1H NMR and micro-
analytical data in accord with the proposed structures: e.g. [EuL2]: m/z
systems, in which longer wavelength excitation is of course
required and where changes in the form of the lanthanide
emission are desirable.
(ESMS2) 847.1660, (C29H39EuN6O14 2H) requires 847.1659. L1a
:
Found: C, 39.2, H, 6.34; N, 12.3. Calc.(as bisammonium salt dihydrate):
C, 39.3; H, 5.93; N, 12.2%. [TbL2]: m/z (ESMS2) 853.1692;
(C29H39TbN6O14 –H) requires 853.1699.
We thank BBSRC for support.
13 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker, L.
Royle, A. S. de Sousa, J. A. G. Williams and M. Woods, J. Chem. Soc.,
Perkin Trans. 2., 1999, 493.
Notes and references
1 A. P. de Silva, D. B. Fox, A. J. M. Huxley, N. D. McClenaghan and J.
Roiron, Coord. Chem. Rev., 1999, 185–186, 297; for examples of Na/K
Communication b000283f
474
Chem. Commun., 2000, 473–474