A long-wavelength fluorescent chemodosimeter selective for Cu(II) ion in water

Full text of DOI:10.1021/ja971221g

7386
J. Am. Chem. Soc. 1997, 119, 7386-7387
Communications to the Editor
Scheme 1
Scheme 2
A Long-Wavelength Fluorescent Chemodosimeter
Selective for Cu(II) Ion in Water
Virginie Dujols,† Francis Ford, and Anthony W. Czarnik*,‡
Department of Chemistry
The Ohio State UniVersity
Columbus, Ohio 43210
ReceiVed April 17, 1997
Chemodosimeters are devices, molecule-sized or larger, that
utilize abiotic receptors to achieve analyte recognition with
concomitant irreversible transduction of a human-observable
signal. In fluorescent chemodosimeters, that signal is fluores-
cence. The properties of ideal fluorescent chemodosimeters are
similar to those of fluorescent chemosensors,1 except that while
real-time response remains desirable, that response reflects a
cumulative exposure to analyte and is therefore not reversible.
In this paper, we report the design and synthesis of a new
fluorescent chemodosimeter for Cu(II) ion in water that
demonstrates usefully large selectivity and signal strength at a
rhodamine-like emission wavelength.
Cu(II) ion displays very high affinities for various polyaza
ligands (e.g., Keq ) 6.3 × 1024 M-1 toward cyclen at pH 72),
but unfortunately a variety of other transition metal ions also
display affinities only somewhat lower. However, the Cu(II)
ion has been known for almost 50 years to promote the
hydrolysis of R-amino acid esters (1) at rates much greater than
those of other metal ions (Scheme 1).3 A key feature of this
reaction is the intermediacy of chelate 2. For most R-amino
acid esters, hydrolysis is complete within seconds at room
temperature and neutral pH under reasonable reactant concentra-
tions, yielding the Cu(II)‚R-amino acid chelate (3; often 1:2
stoichiometry) as product. Selectivity based on time, rather than
on bound/free ratio, is ultimately easier to reduce to practice
for dosimetry applications, as higher concentrations of the
indicator (leading to larger signals) can be used without fear of
saturation under conditions of competing analytes.
POCl3 followed without purification by hydrazine,4 is a color-
less, nonfluorescent substance after crystallization from aceto-
nitrile/water. We hypothesized that the hydrazide group of
compound 4 would provide recognition for the Cu(II) by analogy
to its behavior with R-amino esters. Hydrazides, hydroxamic
acids, and O-acyl hydroxylamines are all known to bind Cu(II)
thusly with resulting enhanced transacylation reactivity.5 Upon
addition of Cu(OAc)2 to a colorless solution of hydrazide 4 in
acetonitrile, both the pink color and fluorescence characteristic
of rhodamine B appear instantly. Because both disappear upon
addition of the chelating ligand cyclen (excess), we propose
that Cu(II) in acetonitrile induces a 4 a 6 equilibrium in much
the same way that the proton induces an analogous rhodamine
B equilibrium in water.
We envisioned that a similar molecular recognition/reactivity
motif might be incorporated into a fluorophore derivative, such
that Cu(II) complexation would lead to a fluorescence increase.
Our approach is depicted in Scheme 2. Rhodamine B hydrazide
(4), prepared in 80% yield by the reaction of rhodamine B with
The conditions of this reaction can be chosen to yield high
selectivity for the Cu(II) ion. A solution of chemodosimeter 4
(0.5 µM) was prepared at pH 7 (0.01 M HEPES buffer), 100
equiv of various metal salts were then added, and the fluores-
cence intensity was monitored with time to produce the results
shown. The salts tested were Ag(I), Al(III), Ca(II), Cd(II), Co-
(II), Cr(III), Cu(II), Eu(III), Fe(III), Ga(III), Gd(III), Hg(II), In-
(III), K(I), Li(I), Mg(II), Mn(II), Na(I), Ni(II), Pb(II), Rb(I),
Sn(IV), Sr(II), U(IV), Yb(III), and Zn(II). After 1 h, only Cu-
(II) and Hg(II) showed a change in UV-visible absorption or
fluorescence. Optimization of assay conditions requires 20%
acetonitrile in pH 7 HEPES buffer. Under these conditions,
the reaction of 0.5 µM 4 with 50 µM Cu(II) was complete within
1 min. The analogous reaction with Hg(II) requires 50 h to
achieve completion. Both reactions afford increases in both
fluorescence and absorbance, such that both fluorimetric and
colorimetric analyses may be obtained.
† Current address: Sensors for Medicine and Science, Germantown, MD
20874. (Email: vdujols@s4ms.com.)
‡ Current address: IRORI Quantum Microchemistry, La Jolla, CA 92037-
1030. (Email: aczarnik@irori.com.)
(1) Czarnik, A. W. Chem. Biol. 1995, 2, 423. Available online at http:
//BioMedNet.com/fluoro. Fluorescent chemosensors for the Cu(II) ion have
been described: Chemosensors of Ion and Molecular Recognition; Des-
vergne, J.-P., Czarnik, A. W., Eds.; NATO ASI Series, Series C: Vol. 492;
Kluwer Academic Press: Dordrecht, 1997; chapters by L. Fabbrizzi, p 75;
A. Czarnik, p 189; and F. Fages, p 221.
(2) Mutsuo, K. J. Chem. Soc., Chem. Commun. 1975, 326.
(3) (a) Kroll, H. J. Am. Chem. Soc. 1952, 74, 2034; Ibid. (b) 2036. (c)
Bender, M. L.; Turnquist, B. W. J. Am. Chem. Soc. 1957, 79, 1889. The
original discovery was made in the 1940s as part of a Department of Defense
program, explaining why publication was delayed.
1
(4) Rhodamine B hydrazide (4): mp 190 °C dec; H NMR (CDCl3) δ
1.16 (t, 12, NCH2CH3), 3.34 (q, 8, NCH2CH3), 3.61 (bs, 2, NH2), 6.29 (dd,
2, xanthene-H), 6.42 (d, 2, xanthene-H), 6.48 (d, 2, xanthene-H), 7.11 (m,
1, Ar-H), 7.45 (m, 2, Ar-H), 7.94 (m, 1, Ar-H); 13C NMR (CDCl3) δ 12.5,
44.25, 65.75, 97.85, 104.45, 107.9, 122.8, 123.7, 127.90, 127.94, 129.9,
132.3, 148.7, 151.4, 153.7, 165.9; EI mass spectrometry, m/e 456.2526,
100% (M)+; M+, calcd 456.2525. Anal. Calcd for C28H32N4O2: C, 73.66;
H, 7.06; N, 12.27; O, 7.01. Found: C, 73.61; H, 7.11; N, 12.34. Full
experimental details can be found in the Supporting Information. Because
this compound forms a stable hydrazone with acetone (fully characterized;
mp 240-245 °C), the isomeric 6-membered ring alternative can be excluded.
The reaction with Cu(II) in water (as compared to acetonitrile
as described above) predictably5 effects a redox hydrolysis of
(5) For lead references, see: Wathen, S. P.; Czarnik, A. W. J. Org. Chem.
1992, 57, 6129.
S0002-7863(97)01221-3 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 31, 1997 7387
Figure 1. An assay for Cu2+, 0-2 µM. Hydrazide 4 (10 µM) in 0.01
M HEPES buffer (pH 7) with 20% (v/v) added CH3CN. Fluorescence
was recorded every 2 min after adding Cu2+: Excitation wavelength
(λex), 510 nm; slit width, 10 nm; emission wavelength (λem), 578 nm;
slit width, 20 nm.
Figure 2. An assay for Cu2+, 0-0.1 µM. Hydrazide 4 (10 µM) in
0.01 M HEPES buffer (pH 7) with 20% (v/v) added CH3CN.
Fluorescence was recorded every 2 min after adding Cu2+: excitation
wavelength (λex), 510 nm; slit width, 10 nm; emission wavelength (λem),
578 nm; slit width, 20 nm.
In summary, we have designed and created a compound with
selectivity for Cu(II) in water based upon that ion’s known
reactivity toward carboxylate derivatives. While the unique
propensity of Cu(II) to promote hydrolysis has been known for
nearly 50 years, this information has not been used previously
for Cu(II) ion quantitation in water. Because of the simplicity
of the analysis, we believe the method may find application in
a variety of settings requiring rapid and accurate Cu(II) ion
analysis.
6 leading to rhodamine B (7) itself in a stoichiometric6 process.
The formation of rhodamine B as product is confirmed by
comparison of the product’s TLC data (three-solvent systems),
electrospray mass spectrometry data, and absorption and emis-
sion properties with those of authentic rhodamine B.7 Addition
of Cu(II) chelating agents, such as EDTA, does not decrease
the intensity of the UV-visible absorption or fluorescence
signals, confirming the irreversible character of this process.
The resulting dosimetric analysis of Cu(II) can be used to
measure Cu(II) ion concentrations in water. Figure 1 depicts
such a titration, with linear response to 2 µM Cu(II) in water.
Figure 2 shows the result of a titration to 0.1 µM Cu(II). Each
reaction used to compose Figures 1 and 2 (including that for
10 nM Cu(II)) was effectively complete within 1 min of reagent
mixing; 2 min were used.8
Acknowledgment. We thank Professors David Hart and Daniel
Leussing for helpful advice during the course of this work and Professor
Matthew Platz for use of a fluorimeter. Support from The Ohio State
University is gratefully acknowledged.
Supporting Information Available: Experimental Details
(3 pages). See any current masthead page for ordering and
Internet access instructions.
(6) While stoichiometric, the exact stoichiometry is nonintegral and
dependent on hydrazide concentration. Experiments designed to understand
the origin of this variable stoichiometry have not led to a defendable reaction
mechanism; thus, neither mechanism nor stoichiometry are depicted or
implied in Scheme 2. Furthermore, less than 1 equiv of rhodamine B is
formed after complete reaction. The empirical observation is that at a given
concentration of hydrazide, the fluorescence and UV intensity increases
are proportional to Cu(II) concentration, permitting the creation of linear
standard curves.
JA971221G
(8) The solutions of Cu(II) were prepared using CuSO4 in double-distilled
water. Various Cu(II) solutions (10-5, 10-4, and 10-3 M) were prepared
separately in volumetric flasks. Solutions of rhodamine B hydrazide were
prepared in acetonitrile by serial dilution. Aqueous solutions (containing
20% acetonitrile) of chemodosimeter 4 were prepared by adding the
acetonitrile solution with stirring to HEPES (0.01 M) at pH 7, with a final
concentration of chemodosimeter 4 equal to 10 µM. Cu(II) assays using
compound 4 were run in a polystyrene cuvette. Aliquots of Cu(II) were
added every 2 min and the fluorescence was recorded.
(7) For details of this and all other aspects of this study, see: Dujols, V.
Ph.D. thesis, The Ohio State University, 1996.