Derivatives of 8-hydroxy-2-methylquinoline are powerful prototypes for zinc sensors in biological systems

Full text of DOI:10.1021/ja0039839

5160
J. Am. Chem. Soc. 2001, 123, 5160-5161
Derivatives of 8-Hydroxy-2-methylquinoline Are
Powerful Prototypes for Zinc Sensors in Biological
Systems
Table 1. Relevant Spectroscopic Data for Oxn Derivatives that
Form Fluorescent Complexes with Zn2
+
derivative
λ
ex, nm
λ
em, nm
ꢀ, cm M-1
a
-1
φa,b
1
2
4
5
7
8
9
353
425
355
356
354
353
369
369
533
460
515
493
499
464
465
535
2286
14200
3796
3433
5563
4564
4826
6061
0.004
<0.001
0.058
0.33
0.24
0.70
†
†
§
Dierdre A. Pearce, Nathalie Jotterand, Isaac S. Carrico, and
Barbara Imperiali*,†
Department of Chemistry
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
0.44
0.10
1
0
a
ReceiVed NoVember 16, 2000
Spectra acquired in 150 mM NaCl, 50 mM HEPES, pH 7.01, 25
b
°
2
C, with 1.00 µM 1-10 and 0.5-1.0 mM ZnCl . Excitation of all
The recent emphasis on understanding the myriad roles of zinc
species provided at λ (355-425 nm), 5 nm slit widths. Quantum
max
1
10
in both normal and diseased cells and tissues has placed an ever
yields were calculated with reference to a quinine sulfate standard.
increasing demand on methods for sensitive and selective methods
for real-time monitoring of free Zn2+ in complex biological
procedures involving the preparation of the corresponding sulfonyl
chloride derivatives using chlorosulfonic acid,8 followed by
addition of these intermediates to excess amine in THF.
samples. Chelation-enhanced fluorescent sensors for zinc, based
2
3
4
on fluorophores such as quinoline, dansyl, fluorescein, and
5
anthracene, have been reported. While each of these agents has
unique advantages, there remain issues with sensitivity, selectivity,
and specificity that may be addressable with an alternate chro-
mophore that is readily amenable to synthetic manipulation.
Herein we report the systematic chemical modification of the
8
-hydroxy-2-methylquinoline (Oxn) unit as a building block for
the development of new sensors employing chelation-enhanced
fluorescence. In particular, improvements in quantum yield from
0
.004 to 0.70 and stepwise blue shifts in fluorescence emission
wavelengths (to a total of over 70 nm) are reported.
A selection of substituted quinoline derivatives bearing the
electron-withdrawing nitro, sulfonic acid, and sulfonamide sub-
stituents (1-9) were prepared and screened for fluorescence
response to Zn2 under pseudobiological conditions. Derivatives
1
+
6
Chemical substitution dramatically influenced the efficiency
of both the absorption and emission properties of the Oxn
derivatives. Table 1 lists relevant extinction coefficients and
7
-6 were prepared by previously described methods. The new
sulfonamide derivatives, 7-9, were prepared by simple two-step
2
+
*
To whom correspondence should be addressed.
quantum yields of the fluorescent Zn -bound derivatives.
Substitution of nitro groups on the Oxn core resulted in
†
Massachusetts Institute of Technology.
§
Present address: Department of Chemistry and Chemical Engineering,
2+
significantly less fluorescent complexes with Zn ; complexes
California Institute of Technology, Pasadena, CA 91125.
(
1) (a) Kimura, E.; Kikuta, E. J. Bioinorg. Chem. 2000, 5, 139-155. (b)
of 2 are weakly fluorescent, and those of 3 and 6 exhibited no
measurable fluorescence in neutral solution. By contrast, deriva-
tization with sulfonic acid or sulfonamide groups resulted in
dramatically enhanced fluorescence properties. The improvement
in quantum yield of the sulfonamide derivatives is particularly
striking; that of 8 is 175 times greater than that of 1. Although
the excitation maxima for the compounds remained essentially
Cox, E. H.; McLendon, G. L. Curr. Opin. Chem. Biol. 2000, 4, 162-165. (c)
Ramboarina, S.; Moreller, N.; Fournie-Zaluski, M.-C.; Roques, B. P.
Biochemistry 1999, 38, 9600-9607.
(
2) (a) Savage, D. D.; Montano, C. Y.; Kasarskis, E. J. Brain Res. 1989,
4
1
96, 257-267. (b) Budde, T.; Minta, A.; White, J. A.; Kay, A. R. Neuroscience
997, 79, 347-358. (c) Nasir, M. S.; Fahrni, C. J.; Suhy, D. A.; Kolodsick,
K. J.; Singer, C. P.; O’Halloran, T. V. J. Biol. Inorg. Chem. 1999, 4, 775-
7
83. (d) Zalewski, P. D.; Forbes, I. J.; Seamark, R. F.; Borlinghaus, R.; Betts,
W. H.; Lincoln, S. F.; Ward, A. D. Chem. Biol. 1994, 1, 153-161.
3) (a) Koike, T.; Watanabe, T.; Aoki, S.; Kimura, E.; Shiro, M. J. Am.
2+
unchanged, the emission wavelengths of the Zn -bound deriva-
(
2+
tives were blue shifted compared to that from Zn -bound 1. The
Chem. Soc. 1996, 118, 12696-12703. (b) Prodi, L.; Bolletta, F.; Montalti,
M.; Zaccheroni, N. Eur. J. Inorg. Chem. 1999, 455-460. (c) Thompson, R.
B.; Maliwal, B. P.; Feliccia, V. L.; Fierke, C. A.; McCall, K. Anal. Chem.
extent of blue shift was dependent on both the type (sulfonamide
derivatives were more blue shifted than sulfonic acid derivatives)
and degree (5,7-disubstituted derivatives were more blue shifted
1
998, 70, 4717-4723.
(
4) (a) Hirano, T.; Kikuchi, K.; Urano, Y.; Higushi, T.; Nagano, T. J. Am.
9
than 5-subsituted derivatives) of substitution (Figure 1a).
Chem. Soc. 2000, 122, 12399-12400. (b) Walkup, G. K.; Burdette, S. C.;
Lippard, S. J.; Tsien, R. Y. J. Am. Chem. Soc. 2000, 122, 5644-5645.
Fluorescence emission properties may be compared using a
sensitivity index based on the product of the quantum yield and
the extinction coefficient at λex. A complete survey of the zinc
complexes (Figure 1b) reveals the additive effect of substitution
(
5) Akkaya, E. U.; Huston, M E.; Czarnik, A. W. J. Am. Chem. Soc. 1990,
1
12, 3590-3593.
(
6) (a) The fluorescence properties of metal complexes of a related species
have been used for detection of metal ions such as zinc following chromato-
graphic separation. Soroka, K.; Vithanage, R. S.; Phillips, D. A.; Walker, B.;
Dasgupta, P. K. Anal. Chem. 1987, 59, 629-636. Dickens, J. E.; Sepaniak,
M. J. J. Microcolumn Sep. 1999, 11, 45-51. (b) Nitro-substituted quinoline
units have been reported as potential chromophoric or fluorophoric sensors.
Prodi, L.; Bargossi, C.; Montalti, M.; Zaccheroni, N.; Su, N.; Bradshaw, J.
S.; Izatt, R. M.; Savage, P. B. J. Am. Chem. Soc. 2000, 122, 6769-6770 and
references therein.
(8) Clarke, H. T.; Babcock, G. S.; Murray, T. F. Benzenesulfonyl Chloride.
In Organic Syntheses, 2nd ed.; Blatt, A. H., Gilman, H., Eds.; John Wiley
and Sons: New York, 1961; Vol. 1, pp 85-87.
(9) Similar blue shifts in emission spectra of zinc complexes of a related
quinoline derivative in methylene chloride have been reported in the literature.
Hopkins, T. A.; Meerholz, K.; Shaheen, S.; Anderson, M. L.; Schmidt, A.;
Kippelen, B.; Padias, A. B.; Hall, H. K. J.; Peyghambarian, N.; Armstrong,
N. R. Chem. Mater. 1996, 8, 344-351. These authors postulate that blue shifts
in emission wavelengths are due to increases in the energy difference between
the highest occupied molecular orbital (HOMO) and the lowest unoccupied
molecular orbital (LUMO), and are generated by introducing electron-
withdrawing groups to the quinoline core. Crystals of the zinc complex of 8
exhibit an intense pale blue fluorescence when irradiated at 365 nm.
(
7) Oxn derivatives prepared and screened in this study: Derivative 1:
Aldrich. 2, 3: Petrow, V.; Sturgeon, B. J. Chem. Soc. 1954, 570-574. 4:
prepared by hydrolysis of 5-sulfonyl chloride of 1, characterization consistent
with literature: Gershon, H.; McNeil, M. E.; Grefig, A. T. J. Org. Chem.
1
1
969, 34, 3268-3270. 5, 6: Gershon, H.; McNeil, M. W. J. Heterocycl. Chem.
972, 9, 659-667. 7-9: prepared by addition of corresponding sulfonyl
chloride to excess amine in THF. 10: Molecular Probes.
1
0.1021/ja0039839 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/02/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5161
Figure 1. (a) Blue shift in normalized fluorescence emission spectra of
Figure 2. Fluorescence microscope images of NIH/3T3 mouse fibroblasts
labeled with 9 in metal free Hank’s balanced salt solution. (a) Wide field
view of cells loaded with 9 (all cells take up the ligand). (b) Image
acquired at higher magnification of cells showing nonuniform “punctate”
patterning of intracellular fluorescence following incubation with 9 (10
µM, 45 min, 21 °C).
4
, 5, 7, and 8 compared to that of 1. The baseline spectrum is that of 8
2
+
in the absence of zinc. (b) Sensitivity index of Zn -bound Oxn
2
+
derivatives, compared to Zn -bound TSQ. Data are normalized relative
to the parent Oxn, 1 (1.00). Experimental conditions are described in
Table 1.
around the Oxn core on the fluorescence signal from the resulting
zinc complexes. The sensitivity of the compounds presented in
this study is compared with that of 6-methoxy-8-(p-toluene-
sulfonamido)quinoline (TSQ), 10, a well characterized intra-
cellular, biochemical, and analytical fluorescence indicator for
zinc that exhibits spectroscopic properties representative of a class
of 8-(p-toluenesulfonamido)quinoline indicators, including Zin-
quin.2 The sensitivity indices of derivatives 5, 7, and 8 were
superior to those of 10, indicating that sensors prepared using
these species might provide a useful fluorescence signal on
considerably brighter than the surrounding cellular material
(Figure 2b). Recent reports have described similar “punctate”
staining with other fluorescent staining agents for intracellular
2+
Zn in fibroblast cell lines and attributed this feature to the
existence of intracellular vesicles containing elevated concentra-
2+ 2c,d
tions of Zn . It is likely that the nonuniform staining of cells
2+
by 9 results from a similar intracellular distribution of Zn . The
,11
punctate fluorescence staining was reversed on incubation with
2
0 µM (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN),
2d
a membrane permeable metal chelating agent, and not reversed
binding Zn2 in biological or near-neutral environmental samples.
+
12
by incubation with 20 µM EDTA (which does not transport
The quantum yield of the fluorophores is the result of contribu-
tions from a number of sources, including molecular dipole,
resonance structures, and solvent-fluorophore interactions, and
further study will be required to describe the cumulative nature
of these contributions.
To examine whether sulfonamide derivatives such as 7 and 8
would, in fact, prove useful components of zinc chemosensors,
the more hydrophobic derivative 9 was prepared for intracellular
experiments.13 Fluorescent labeling of mouse fibroblasts (NIH/
2c
through the cell membrane). Consequently, the observed fluo-
rescence from 9 can be said to result from metal-bound probe
located within the cell.
In summary, the quantum yields of new derivatives 7-9, when
2+
complexed to Zn in pseudobiological conditions, are superior
to quinoline derived probes reported in the literature. The affinity
of these simple species for zinc may make them useful probes of
2b,14
cellular events associated with elevated zinc levels.
Moreover,
these new derivatives are very good candidates for subsequent
3T3) occurred after incubating the cells with 9 (Figure 2a). The
15
incorporation into sensor designs with an extended superstructure
fluorescence within the cell is nonuniform; some regions appear
for sensitive and selective detection of zinc in biological and
environmental applications, studies of which are in progress.
(
10) (a) Chen, R. F.; Kernohan, J. C. J. Biol. Chem. 1967, 242, 5813-
823. (b) Chen, R. F. Anal. Biochem. 1967, 19, 374-387.
11) (a) Compano, R.; Ferrer, R.; Guiteras, J.; Prat, M. D. Analyst 1994,
19, 1225-1228. (b) Nowicki, J. L.; Johnson, K. S.; Coale, K. H.; Elrod, V.
5
(
Acknowledgment. This research was supported by grants from the
NSF (CHE-9412442) in the initial phases of this project and continued
support from the NIH (GM-58716) and a postdoctoral fellowship to N.J.
from the Swiss National Science Foundation. We thank V. Goncharov
for technical assistance and advice with preparation of cells for loading
and Professor L. Griffith and B. Harms for helpful discussions.
1
A.; Lieberman, S. H. Anal. Chem. 1994, 66, 2732-2738.
(
12) Job’s plot analyses indicate that at pH 7.0 a mixture of 1:1 and 2:1
2+
complexes of Zn(II) is formed. Half-maximal intensity is reached at a [Zn
]
free
of 6.0 and 27 µM for 7 and 8, respectively. This compares well with 8-(p-
toluenesulfonamido)quinoline indicators such as TSQ and Zinquin (ligands
with K 1-20 µM), ref 2, and references therein. Cell permeable indicators
d
2+
with higher affinity for Zn have been reported in the literature, including
refs 2c and 4.
Supporting Information Available: Experimental details, including
synthetic procedures, relevant spectra, pH profiles, and metal ion
selectivity of 9, determination of quantum yields, and imaging of probe-
loaded cells (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
+
+
(
13) Of the biologically relevant metal ions, Cu2+, Fe3+, Na , and K do
2+
not form fluorescent complexes with the ligands. Only Cu , and to a lesser
extent Fe3 , displaced Zn from the corresponding complexes. Levels of both
species, particularly copper, are highly regulated in cells (Rae, T. D.; Schmidt,
P. J.; Pufahl, R. A.; Culotta, V. C.; O’Halloran, T. V. Science 1999, 284,
+
2+
2
+
2+
8
05-808). Addition of Ca and Mg resulted in weak fluorescence
JA0039839
2
+
responses. Fluorescence emission from 9, either in the apo form or the Zn
complex, was essentially independent of pH in the pH 4-8 range. The affinity
(14) (a) Frederikson, C. J.; Suh, S. W.; Silva, D.; Frederickson, C. J.;
Thompson, R. B. J. Nutr. 2000, 130, 1471S-1483S. (b) Cole, T. B.; Wenzel,
H. J.; Kafer, K. E.; Schwartzkroin, P. A.; Palmiter, R. D. Proc. Natl. Acal.
Sci. 1999, 96, 1716-1721.
(15) Walkup, G. K.; and Imperiali, B. J. Org. Chem. 1998, 63, 6727-
6731.
of 9 for zinc is similar to that of 8; low solubility of complexes of 9 made it
d
difficult to obtain a precise value for an apparent K . Imaging experiments
were performed with either the presence or absence of 9 in the media. In the
second instance, some loss of intracellular fluorescence intensity was observed
after an extended period of observation (ca. 45 min).