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DPA-substituted coumarins as chemosensors for zinc(II): modulation of

the chemosensory characteristics by variation of the position of the
chelate on the coumarin
Nathaniel C. Lim and Christian Brückner*
Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA.
E-mail: [email protected]
Published on 01 April 2004. Downloaded by Lomonosov Moscow State University on 07/12/2013 12:50:03.

Received (in West Lafayette, IN, USA) 5th March 2004, Accepted 22nd March 2004
First published as an Advance Article on the web 1st April 2004

The sensory capabilities of two novel di(2-picolyl)amine (DPA)- Relative fluorescence emission intensities (Iemission) observed in
substituted coumarins are described and it is shown that the cells stained with a zinc-specific chemosensor can only be
variation of the point of attachment of the DPA group to the correlated with relative increases in [Zn2+], but measurement of an
coumarin framework controls their sensing behavior: the absolute Iemission does not allow the determination of an absolute
4-substituted system is a CHEF-type sensor that shows a [Zn2+]. In part, this is because f of any fluor is solvent dependent,
significant increase in fluorescence intensity upon Zn2+ binding, but the solvent properties of the local environments in which the
whereas the 3-substituted system is a ratiometric sensor. sensors accumulate are not known. The measurement of absolute
[Zn2+] can be achieved, however, with a ratiometric sensor.8 A
The development of metal ion chemosensors in general, and for ratiometric probe responds upon binding to an analyte with a shift
Zn2+ in particular, has received considerable attention.1,2 The use of in its lmax
emission. This shift should be large enough to allow the
Zn2+-specific chemosensors in biological systems promises to close determination of the intensity ratio of the signals for co-existing
the knowledge gap between the well-defined structural bio- Zn2+-free and Zn2+-bound species. Together with the known Kd of
chemistry of zinc and the understanding of zinc homeostasis and the sensor, this allows the determination of [Zn2+].8
action.3–7 Ratiometric sensing behavior can be expected when the binding
We recently reported a coumarin–cyclen-based chemosensor for of the analyte changes the electronic properties of the chromophore,
Zn2+.5 Though this sensor proved capable of imaging Zn2+ in live but the realization of this is non-trivial, as recent examples have
cells, its binding kinetics was slow and the fluorescence intensity shown.4a,7 The lactone oxygen in coumarins is a potential donor
increase upon Zn2+ binding was a modest 4.4-fold. We report here atom attached to the chromophore, but steric restraints prevent the
the synthesis of coumarin–DPA-based chemosensors with much lactone oxygen in sensor 3 from participating in the coordination
improved sensory characteristics, including the realization of a event. However, moving the attachment point for the chelating
long-coveted ratiometric sensor for Zn2+.4a,7 moiety on the coumarin from the 4- to the 3-position potentially
Nucleophilic substitution of 4-bromomethyl-6,7-dimethoxycou- allows for carbonyl participation. We therefore synthesized the
marin (1) with DPA (2) produces sensor assembly 3 (Scheme 1).† sensor assembly 4 by reductive amination of coumarin aldehyde 59
DPA has proven its utility in the design of chemosensors for Zn2+.4 with DPA (Scheme 1).†
In addition, as an open chain chelate, it promised to alleviate the This particular 7-amino-derivatized coumarin derivative was
slow binding kinetics of our analogously constructed cyclen-based chosen because it features longer lmax max
excitation/lemission than 6,7-dime-
sensor.5
Fig. 1(A) shows the excellent chemosensory response of sensor
3. Addition of 1 equiv. Zn2+ increases the integrated fluorescence
intensity 23-fold. This chelation-enhanced fluorescence (CHEF)
increase compares favorably to those of most known zinc-specific
chemosensors.6 The fluorescence quantum yields, f, for 3 and
3·Zn2+ in MeOH are 0.038 and 0.88, respectively (e for 3 and
3·Zn2+ in MeOH are 7600 and 6800 cm21 M21, respectively). As
expected, the binding of 3 to Zn2+ is fast and completed upon
mixing of ligand and metal. Based on a Hill plot analysis, sensor 3
forms a 1:1 complex with Zn2+ and is suitable for measurements in
aqueous media of pH 4–11 [Fig. 1(B)]. The Kd of the complex, Fig. 1 (A) Emission spectra of 3 (—) and 3 + 1 equiv. Zn2+ (Ã). (B) pH-
measured by titration of 3 with Zn2+ in MeOH, is of the order of 0.5 dependent fluorescence profile of 3. Conditions: [3] = 192 mM in MeOH
mM. This value is in accord with those found for other DPA-based (A) and 10 mM in water containing 1 mM KOH and 100 mM KCl, pH
sensors.4 adjusted with HCl (B); 25 °C; lexcitation = 343 nm.
DOI: 10.1039/b403448a

Scheme 1 Reagents and conditions: (i) CH2Cl2, Na2CO3, r.t., 1 day (90% yield); (ii) ClCH2CH2Cl, NaBH(OAc)3, r.t., 1 day (92% yield).†

1094 Chem. Commun., 2004, 1094–1095 This journal is © The Royal Society of Chemistry 2004
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thoxycoumarin (lmaxexcitation = 400 nm for 4, as compared to 343 nm mimicking the presence of Zn2+. As is observed for most
for 3). The use of longer wavelengths has a number of practical chemosensors for Zn2+, Cd2+ binds strongly to the sensor. For
advantages in (confocal) fluorescence microscopy. sensor 4, Cd2+ also elicits the same ratiometric response as Zn2+.
The results of a spectrophotometric titration of 4 with Zn2+ are However, the concentration of Cd2+ in healthy cells is low. Thus, in
shown in Fig. 2. The 31 nm shift of lmax
absorption upon addition of Zn
2+ practice, this ion will not interfere with the measurement of Zn2+ in
demonstrates the Zn2+-induced perturbation of the electronic live cells. The results of the biological evaluation of the sensors will
structure of the chromophore. Thus, a ratiometric fluorescence be published in due course.
response can be expected and, indeed, is observed. Incremental In conclusion, we have synthesized novel chemosensors for
additions of Zn2+ result in a 21 nm bathochromic shift of lmax emission zinc(II) and have refined the design paradigms for coumarin-based
Published on 01 April 2004. Downloaded by Lomonosov Moscow State University on 07/12/2013 12:50:03.

of 4. This shift is solvent dependent and is minimized in solvent CHEF-type and ratiometric sensors.
systems containing increasing amounts of water. We suggest the This work was supported by the UConn Research Foundation
structure shown in Scheme 1 for 4·Zn2+. The fourth coordination and the Petroleum Research Fund (PRF-37432-G1), administered
site of the tetrahedrally N3O-coordinated metal center is provided by the American Chemical Society.
by the lactone oxygen, although the formation of a pentacoordinate
metal center by inclusion of a water or alcohol molecule cannot be
excluded.3a The solvent dependency of the lmax emission shift suggests
that water is successfully competing with the carbonyl oxygen for
coordination to the metal center, resulting in degradation of the Notes and references
ratiometric response. † Selected experimental data for 3: 1H NMR (CDCl3, 400 MHz): d 8.54 (d,
In stark contrast to sensor 3, 4 exhibits only minimal CHEF-type J = 4.1 Hz, 2H), 7.68–7.64 (m, 2H), 7.45 (d, J = 7.8 Hz, 2H), 7.31 (s, 1H),
behavior. Sensor 4 in its free-base form is already ‘switched on’, 7.20–7.17 (m, 2H), 6.82 (s, 1H), 6.60 (s, 1H), 3.94 (s, 3H), 3.90 (s, 9H) ppm;
13C NMR (CDCl , 100 MHz): d 161.8, 158.7, 153.2, 152.9, 149.9, 149.4,
with a f of 0.64 (e = 16 900 cm21 M21). Thus, a methyleneamino 3
146.2, 136.9, 123.4, 122.6, 112.4, 111.5, 106.0, 100.1, 60.9, 56.7, 56.5, 55.7
group attached to the 3-position of the coumarin does not quench
ppm; HR-MS (FAB+ of MH+, NBA): m/z calcd for C24H24N3O4 418.4744,
the fluorescence of free-base 4 effectively; therefore, chelation found 418.4737. For 4: 1H NMR (CDCl3, 400 MHz): d 8.52 (d, J = 4.8 Hz,
results only in a minimal increase in the fluorescence intensity. 2H), 7.71 (s, 1H), 7.66–7.64 (m, 4H), 7.15–7.11 (m, 2H), 6.87 (s, 1H), 3.92
This, however, is not a disadvantage for the construction of (s, 4H), 3.66 (s, 2H), 3.25 (q, J = 5.5 Hz, 4H), 2.88 (t, J = 6.4 Hz, 2H), 2.76
ratiometric sensors. (t, J = 6.4 Hz, 2H), 1.99–1.96 (m, 4H) ppm; 13C NMR (CDCl3, 100 MHz):
The selectivity of sensors 3 and 4 for Zn2+ makes them suitable d 162.8, 159.5, 151.0, 149.0, 145.4, 141.9, 136.4, 124.8, 122.8, 121.9,
for use in biological systems. Fig. 3 shows the results of an Mn+ 118.2, 117.4, 108.5, 106.4, 60.1, 53.2, 49.9, 49.6, 27.5, 21.5, 20.6, 20.3
binding and competition study of 3 (the profile of 4 is very similar ppm; HR-MS (FAB+ of MH+, NBA): m/z calcd for C28H29N4O2 453.5666,
to that of 3). While a range of metals bind to the sensor, the addition found 453.5658.
of 1 equiv. Zn2+ outcompetes most. The paramagnetic ions Ni2+ and
Cu2+ remain bound, but due to their fluorescence quenching 1 A. P. de Silva, D. B. Fox, H. J. M. Huxley and T. S. Moody, Coord.
properties, these ions will not provide a false positive signal Chem. Rev., 2000, 205, 41.
2 For representative recent reports of in vitro sensors for zinc, see: (a) T.
Hirano, K. Kikuchi, Y. Urano, T. Higuchi and T. Nagano, Angew. Chem.,
Int. Ed., 2000, 39, 1052; (b) M. C. Kimber, I. B. Mahadevan, S. F.
Lincoln, A. D. Ward and E. R. T. Tiekink, J. Org. Chem., 2000, 65, 8204;
(c) J. C. Loren and J. S. Siegel, Angew. Chem., Int. Ed., 2001, 40, 754; (d)
S. Aoki, S. Kaido, H. Fujioka and E. Kimura, J. Inorg. Chem., 2003, 42,
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Gunnlaugsson, T. C. Lee and R. Parkesh, Org. Biomol. Chem., 2003, 1,
3265.
3 For representative recent reports of sensors for zinc for which biological
data were presented, see: (a) S. C. Burdette, G. K. Walkup, B. Springler,
Fig. 2 (—) UV-vis spectral titration of 4 with Zn2+ (0–1 equiv.). (Ã) R. Y. Tsien and S. J. Lippard, J. Am. Chem. Soc., 2001, 123, 7831; (b) K.
Fluorescence response upon titration of 4 with Zn2+ (0–1 equiv.); lexcitation R. Gee, Z.-L. Zhou, W.-J. Qian and R. Kennedy, J. Am. Chem. Soc., 2002,
= 410 nm. Conditions: [4] = 100 mM in MeOH; 25 °C. 124, 776; (c) S. L. Sensi, D. Ton-That, J. H. Weiss, A. Rothe and K. R.
Gee, Cell Calcium, 2003, 34, 281; ; see also ref. 4, 5 and 7.
4 (a) S. C. Burdette and S. J. Lippard, Inorg. Chem., 2002, 41, 6816; (b) S.
C. Burdette, C. J. Frederickson, W. Bu and S. J. Lippard, J. Am. Chem.
Soc., 2003, 125, 1778.
5 N. C. Lim, L. Yao, H. C. Freake and C. Brückner, Bioorg. Med. Chem.
Lett., 2003, 13, 2251.
6 For sensors with larger fluorescence increases, see, for example: C. J.
Fahrni and T. V. O’Halloran, J. Am. Chem. Soc., 1999, 121, 11 448; see
also ref. 2a.
7 (a) M. Taki, J. L. Wolford and T. V. O’Halloran, J. Am. Chem. Soc.,
2004, 126, 712; (b) C. J. Chang, J. Jaworski, E. M. Nolan, M. Sheng and
S. J. Lippard, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 1129.
8 J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer
Fig. 3 Mn+-selectivity profile of sensor 3: (grey bars) relative integrated Academic/Plenum, New York, 1999.
emission intensity of 3 + 1 equiv. Mn+; (black bars) relative integrated 9 (a) J. Van Gompel and G. B. Schuster, J. Org. Chem., 1987, 52, 1465; (b)
emission intensity of 3 + Mn+, followed by 1 equiv. Zn2+. Conditions: [4] = S. Padmanabhan, R. Peri and D. J. Triggle, Synth. Commun., 1996, 26,
163 mM in MeOH; lexcitation = 343 nm; 25 °C. 827.

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