Photodynamic fluorescent metal ion sensors with parts per billion sensitivity

Article abstract of DOI:10.1021/ja974181p

We report herein the synthesis and spectroscopic study of the first spiropyrandinolines that function as sensors for metal ions in the parts per billion range. These systems operate by either photochemically or chemically induced reversible formation of merocyanine metal ion complexes. The application of this novel photodynamic sensing material to sensor technology is discussed.

Full text of DOI:10.1021/ja974181p

J. Am. Chem. Soc. 1998, 120, 3237-3242
3237
Photodynamic Fluorescent Metal Ion Sensors with Parts per Billion
Sensitivity
Jeffrey D. Winkler,* Corinne M. Bowen, and Veronique Michelet
Contribution from the Department of Chemistry, The UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104
ReceiVed December 8, 1997. ReVised Manuscript ReceiVed February 12, 1998
Abstract: We report herein the synthesis and spectroscopic study of the first spiropyrandinolines that function
as sensors for metal ions in the parts per billion range. These systems operate by either photochemically or
chemically induced reversible formation of merocyanine metal ion complexes. The application of this novel
photodynamic sensing material to sensor technology is discussed.
Introduction
documented.5 While the nonphotolytic thermal equilibrium
between 1 and 2 favors the neutral spiropyran by ca. 2.5 kcal/
mol,6 we have found that incorporation of the spiropyran into
the 19-membered ring lactone 3 leads to a 3 kcal/mol change
in the relative energies of 3 and 4, effectively replacing hν1 in
Scheme 1 with the strain energy of the macrocyclic ring
(Scheme 2).7 Since the development of devices that are
dependent on a single wavelength of light would be inherently
easier to manipulate, we have examined other alternatives to
hν1 for the opening the spiropyran 1 to merocyanine 2.
Several years ago, Taylor and Phillips independently reported
that suitably substituted spiropyrans 5 (R ) Me) could be used
to bind metal ions in the open merocyanine form as shown in
6 (Scheme 3).8 We subsequently established that this binding
is photoreversible and that this system can be used for the
photodynamic transport of metal ions across an organic mem-
brane.9 Photoreversibility of metal ion binding has also been
demonstrated in spiropyran-crown ether conjugates by Inouye
and co-workers.5a It was clear from our preliminary studies
with the 5 f 6 system that a significant increase in sensitivity
(5 f 6 operating in the millimolar metal ion concentration
range) would be required for the application of this strategy to
the synthesis of metal ion sensors. We describe herein the
extension of this spiropyran-based technology to the design and
synthesis of the first highly fluorescent photoreVersible spiro-
pyranindoline “real-time” metal ion sensors with considerably
greater sensitivity (parts per billion range) and lower fatigue
rates than previously described systems.5a
The design and synthesis of functional molecules that could
serve as molecular devices for sensors, switching, and signal
transduction is an area of intense activity and of tremendous
potential significance.1 A key feature for applications involving
sensing is reversibility, i.e. off-rate, in any given recognition
process, a property that also figures prominently in the develop-
ment of efficient switching mechanisms.2
The development of optical methods for the detection and
estimation of clinically and environmentally important species,
such as metal ions, is an important area of contemporary sensor
research.1e-g Cation receptors that lead to changes in fluores-
cence on metal ion binding, which are referred to as fluorescent
chemosensors or fluoroionophores, offer distinct advantages over
those that employ other signaling mechanisms in terms of
sensitivity and selectivity. Several research groups have
developed systems that couple metal cation binding to changes
in fluorescence emission.3,4
The spiropyran nucleus is an attractive starting point in such
constructions as the photoreversibility of such systems is well-
(1) (a) Molecular Electronic DeVices; Carter, F. L., Siatkowski, R. E.,
Wohltjen, H., Eds.; Elsevier: Amsterdam, 1988. (b) Nanotechnology;
Crandall, B. C., Lewis, J., Eds.; MIT Press: Cambridge, MA, 1992. (c)
Drexler, E. Nanosystems: Molecular Machinery, Manufacturing and
Computation; Wiley: New York, 1992. (d) Credi, A.; Balzani, V.; Langford,
S.; Stoddart, J. J. Am. Chem. Soc. 1997, 119, 2679-2681. (e) Czarnik, A.
W. Curr. Biol. 1995, 2, 423-428. (f) Czarnik, A. W. Acc. Chem. Res. 1994,
27, 302-308. (g) Valeur, B.; Bardez, E. Chem. Br. 1995, 216-220. (h)
deSilva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;
McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515-
1566.
Results and Discussion
(2) (a) Wagner, R. W.; Lindsey, J. S.; Seth, J.; Palaniappan, V.; Bocian,
D. J. Am. Chem. Soc. 1996, 118, 3996-3997. (b) Ashton, P.; Ballardini,
R.; Balzani, V. Boyd, S.; Credi, A.; Gandolfi, M.; Gomez-Lopez, M.; Iqbal,
S. Philip, D.; Preece, J. Prodi, L.; Richetts, H.; Stoddart, J.; Tolley, M.;
Venturi, M.; White, A.; Williams, D. Chem. Eur. J. 1997, 3, 152-170.
(3) Fluorescent Chemosensors for Ion and Molecule Recognition;
Czarnik, A. W., Ed.; ACS Symposium Series 538; American Chemical
Society: Washington, DC, 1992.
(4) For examples since 1996, see: (a) Walkup, G.; Imperiali, B. J. Am.
Chem. Soc. 1997, 119, 3443-3450. (b) Diamond, D.; McKervey, M. A.
Chem. Soc. ReV. 1996, 15-24. (c) Ayadim, Habib Jiwan, J. L.; De Silva,
A. P.; Soumillion, J. Tetrahedron Lett. 1996, 7039-7042. (d) Fabbrizzi,
L.; Licchelli, M.; Pallavicini, P.; Sacchi, D.; Taglietti, A. Analyst 1996,
121, 1763-1768. (e) Kubo, K.; Sakurai, T. Chem. Lett. 1996, 959. (f)
Desvergne, J.; Rau, J.; Cherkaoui, O.; Zniber, R.; Bouas-Laurent, H.;
Lahrahar, N.; Meyer, U.; Marsau, P. New J. Chem. 1996, 20, 881-893.
(g) Fages, F.; Bodenant, B.; Weil, T. J. Org. Chem. 1996, 61, 3956-3961.
The first compound that we examined is the 8-hydroxyqui-
nolol-derived spiropyran 11, which was prepared by a modifica-
(5) Bertelson, R. C. In Photochromism; Brown, G. H., Ed.; Wiley: New
York, 1971, and references therein. For recent examples of the utility of
spiropyrans in photoreversible systems, see: (a) Inouye, M.; Akamatus,
K.; Nakazumi, H. J. Am. Chem. Soc. 1997, 119, 9160-9165. (b) Tamaki,
T.; Ichimura, K. J. Chem. Soc., Chem. Commun. 1989, 1477-1479. (c)
Kimura, K.; Yamashita, T.; Yokoyama, M. J. Chem. Soc., Perkin Trans. 2
1992, 613-619.
(6) Flannery, J., Jr. J. Am. Chem. Soc. 1968, 90, 5660-5671.
(7) Winkler, J.; Deshayes, K. J. Am. Chem. Soc. 1987, 109, 2190-2191.
(8) (a) Phillips, J.; Mueller, A.; Przystal, F. J. Am. Chem. Soc. 1965, 87,
4020. (b) Taylor, L.; Nicholson, J.; Davis, R. Tetrahedron Lett. 1967, 1585-
1588.
(9) Winkler, J.; Deshayes, K.; Shao, B. J. Am. Chem. Soc. 1989, 111,
769-770.
S0002-7863(97)04181-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/24/1998

3238 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Winkler et al.
Minimal fluorescence at 610 nm is observed with a 10-5
Scheme 1
M
ethanolic solution of quinolinespiropyranindoline (SP) 11 upon
excitation at 550 nm (ΦF ) 0.0015),13 the absorbance maximum
of the metal-free merocyanine (MC) 13, a result that is consistent
with the predominance of 11 in the absence of exogenous metal
ion or UV light (Scheme 4). Addition of 1 equiv of ZnCl2 to
the solution of 11 and excitation at 572 nm (the absorbance
λmax of chelate 12 in ethanol where M+ ) Zn2+) led to a 14-
fold increase in emission intensity at 610 nm (Figure 1). Similar
effects have been observed with other divalent and trivalent
cations, and our results are summarized in Figure 2.
Scheme 2
This dramatic increase in fluorescence intensity is due not
only to an increase in the concentration of 12 but also to the
coordination of metal ion by the fluorophore. While the
difference in the changes in the long-wavelength absorbance
maxima of an ethanolic solution of 11 upon photochemical
generation of 13 (∆A550 nm ) 0.79)14 and upon addition of 1
equiv of ZnCl2 to 11 to form 12 (∆A572 nm ) 0.52) are small,
the changes in the fluorescence emission intensities are dramatic.
These results are consistent with data reported for metal-bound
and metal-free 8-hydroxyquinoline (8-HQ)15 and have been
recently attributed to the role of the metal cation in inhibiting
photoinduced tautomerization of the 8-hydroxyquinoline moiety
which leads to nonradiative deexcitation.16 Emission in 12
(metal-bound) is enhanced relative to 13 (metal-free) when the
pathways for radiationless relaxation of the excited state
available to 13 are no longer available in the Zn2+-chelated
species 12.
Scheme 3
Scheme 4
These results have been extended to mixed aqueous systems
with similar results, i.e. the sensitivity of 11 for Zn2+ in 1:1
ethanol/water is 3.3 ppb. We have established that this modest
attenuation in sensitivity is due to the decrease of the fluores-
cence of 12 in aqueous media and not due to a decrease in the
denticity of the zwitterion 12 for the metal ion in the presence
of water.
The magnitude of the fluorescence enhancement of 11 in the
presence of Cd2+, another group IIb metal ion, is smaller than
that observed with Zn2+, which could be attributed to the larger
size of Cd2+ relative to Zn2+. Enhanced emission of 11 was
also observed in the presence of certain alkaline earth metals.
Exposure of 11 to MgCl2 results in an increase of fluorescence
intensity of the same magnitude as that observed with Zn2+
.
Since the binding affinities of 8-hydroxyquinoline, the chro-
mophore responsible for metal ion binding in 11, for Mg2+ and
Zn2+ are different,17 the similarity of the resulting emission
enhancements of 11 may be explained by differences in the
stoichiometry or geometry of the respective metal complexes.
The formation of chelates of 12 with other transition metals
(Cu2+, Ni2+, Co2+, and Fe3+) can be monitored by an increase
in the long-wavelength absorbance maxima. For example,
addition of 1 equiv of CuCl2 to an ethanolic solution of 11 results
(11) Fiedler, H. Arch. Pharm. 1964, 297, 108-110.
(12) Hercules, D.; Rogers, L. Spectrochim. Acta 1959, 15, 393-408.
(13) The fluorescence quantum yields of 11 was measured by a relative
method using cresyl violet as a standard, Olmsted, J., III. J. Phys. Chem.
1979, 83, 2581-2584.
tion of the procedure reported by Phillips as outlined in Scheme
4.10 We found it advantageous to perform the ozonolytic
cleavage of 711 on the corresponding benzoate 8 (benzoyl
chloride, pyridine, 51%). Ozonolysis of 8 and reaction of the
crude product with dimethyl sulfide and then ethanolic potas-
sium carbonate gave the 7-formylquinolol (9), in 53% yield over
the two steps. Condensation of 9 with Fisher’s base 1012 gave
the spiropyran 11 (butanone, reflux, 16 h, 57%) as a purple
solid (mp 193 °C).
(14) Przystal, F.; Rudolph, T.; Phillips, J. P. Anal. Chim. Acta 1968, 41,
388-391.
(15) (a) Hercules, D. M.; Rogers, L. B. Spectrochim. Acta 1959, 15,
393-408. (b) Popovych, O.; Rogers, L. B. Spectrochim. Acta 1960, 16,
49-57. (c) Lytle, F. E.; Storey, D. R.; Juricich, M. E. Spectrochim. Acta
1973, 29A, 1357-1369.
(16) Bardez, E.; Devol, I.; Larrey, B.; Valeur, B. J. Phys. Chem. B 1997,
101, 7786-7793. For an earlier rationale based on triplet state theory and
spin-orbit coupling, see: Popovych, O.; Rogers, L. B. Spectrochim. Acta
1959, 584-592.
(17) Perrin, D. D. Stability Constants of Metal-Ion Complexes. Organic
Ligands (IUPAC); Pergamon Press: New York, 1979; Part B, p 648.
(10) Przystal, F.; Phillips, J. P. J. Heterocycl. Chem. 1967, 4, 131-132.

Metal Ion Sensors with Parts per Billion SensitiVity
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3239
Figure 1. Fluorescence emission spectra of spiropyran 11 (10-5 M ethanol, room temperature): (A) emission of “metal-free” solution; (B) emission
after addition of 1 equiv of ZnCl2.
Figure 2. Relative fluorescence emission intensities of 11 (10-5 M ethanol) and 15 (10-5 M in benzene) at room temberature for “metal-free”
solutions and these spiropyrans in the presence of several metal chlorides.
in a blue shift of the absorbance maximum and an increase in
the absorbance value (∆A548 nm ) 0.22). In the Cu2+ chelate
of 12, the proximity of the paramagnetic metal ion to the
unpaired electrons of the ligand leads to spin-orbit coupling
and intersystem crossing, resulting in complete quenching of
fluorescence emission from the 12 chelate (Figure 2). Similar
the metal binding is chemically reversible, it does not establish
the facile photochemical reversibility that was characteristic of
our earlier study with the piperidinomethyl spiropyrans 5 a 6
(Scheme 3).9
We reasoned that the phenolate in 12 (Scheme 4) was a better
ligand for the metal ion than p-nitrophenolate 6 (Scheme 3).
The p-nitro group withdraws electron density from the phenolate
oxygen, decreasing the denticity of 6 and thereby facilitating
reversible binding. We therefore prepared the nitroquino-
linespiropyran 15 (Scheme 5) by condensation of Fisher’s base
10 with 7-formyl-8-hydroxy-5-nitroquinoline (14).11 In contrast
to the modest absorbance bands (554 and 592 nm) that were
observed with ethanolic solution of 11, strong absorbance at
560 nm was observed with an ethanolic solution of the nitro-
effects were observed with Ni2+, Co2+, and Fe3+
.
While irradiation of metal chelate 12 (M+ ) Zn2+) did not
liberate metal ion with regeneration of 11, the liberation of the
metal ion could be achieved chemically, i.e. by addition of 1
equiv of nitrilotriacetic acid, N(CH2CO2H)3, which sequesters
the metal ion, liberating the closed spiropyran 11. The
chemically regenerated 11 can then be recycled for another
metal ion binding event. While this result demonstrates that

3240 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Winkler et al.
Scheme 5
in benzene). The covalent attachment of these metal-ion sensors
to fiber optic surfaces, which could lead to a practically useful
metal-ion sensing device, is currently underway in our labora-
tory, and our results will be reported in due course.
Experimental Section
General Methods. All reagents and solvents were obtained from
Aldrich, Eastman, Fisher, or Sigma. Tetrahydrofuran was distilled from
sodium/benzophenone. Methylene chloride, benzene, and toluene were
distilled from calcium hydride. All reactions were performed under a
dry argon atmosphere unless specified otherwise. Reaction progress
was monitored by analytical thin-layer chromatography (TLC), per-
formed with 0.25-mm Whatman silica gel glass plates containing F-254
indicator. Visualization on TLC was achieved by either UV light (254
nm), phosphomolybdic acid indicator, potassium permanganate, or ceric
sulfate indicator. Flash chromatography was performed with 32-63-
mm silica gel packing. Proton magnetic resonance spectra were
recorded at 500 MHz and carbon spectra were recorded at 125 MHz
on a Bruker AMX-500, with chemical shifts reported as parts per million
downfield using CHCl3 (1H, δ 7.24; 13C, δ 77.0) or C6H6 (1H, δ 7.28;
13C, δ 128.0) as an internal standard. Melting points were determined
using a Thomas-Hoover apparatus and are uncorrected. Infrared spectra
were acquired on a Perkin-Elmer 1600 FT-IR. Neat IR spectra were
acquired using a NaCl plate. High-resolution mass spectra were
obtained at the University of Pennsylvania Mass Spectrometry Service
Center on either a VG micromass 70/70H high-resolution double-
focusing electron impact/chemical ionization spectrometer or a VG
ZAB-E spectrometer. UV/visible absorbance spectra were acquired
on a Hewlett-Packard 8452A diode array spectrophotometer, and
fluorescence emission spectra were acquired on a Photon Technologies
International A1010 luminescence spectrometer. Emission measure-
ments were performed in a four-sided quartz cell at room temperature
and are uncorrected for detector response. Standard solutions (10-3
M) for spectroscopic measurements were prepared using spectropho-
tometric quality solvents (absolute ethanol was obtained from Pharmco)
and volumetric flasks. Appropriate dilutions were carried out using
volumetric pipets.
quinolinespiropyran 15. The introduction of the nitro substituent
shifts the spiropyran a merocyanine equilibrium in polar
solvents such as ethanol to favor the open form 17.18 In less
polar solvents, i.e. benzene or ethyl acetate, the predominant
form is 15. Like spiropyranquinoline 11, the fluorescence
emission of 15 is also sensitive to group IIb and several other
transition metals but inert to alkali metal chlorides (Figure 2).
However, unlike 11, 15 is also relatively inert to alkaline earth
cations.
Addition of 1 equiv of ZnCl2 to a benzene solution of 15
resulted in a 9-fold enhancement of fluorescence emission
(Figure 3). The magnitude of this increase in relative intensity
is less than that observed with 11. Likewise, only a 3-4-fold
increase in emission intensity of 16 was observed upon addition
of 1 equiv of MgCl2 or CdCl2. These differences may be due
simply to the reduced chelating ability of the nitrated hydroxy-
quinoline chromophore. The metal ion binding constants of
5-nitro-8-hydroxyquinoline are typically lower than those of
8-hydroxyquinoline. It is interesting to note that HgCl2 does
not follow the trend exhibited by other group IIb metals. A
nearly 20-fold increase of emission intensity of 16 was observed
upon addition of 1 equiv of HgCl2. Studies to determine the
basis for these observed differences are currently underway.19
We were delighted to find that the ejection of metal ion from
16 occurs upon brief irradiation of the Zn2+ chelate with visible
light (150-W tungsten flood lamp, 30 s to 5 min). Rapid
dissociation of approximately 60% of the fluorescent Zn2+
chelate 16 accompanied by regeneration of 15 occurs with
expulsion of the metal ion (Figure 3). The regenerated
spiropyran is fully functional and has been run through 10 cycles
of metal binding-release without detectable fatigue. Incomplete
photochemical reversal of metal ion binding with 16, in contrast
to the complete reversal observed on exposure of 12 to
nitrilotriacetic acid, may be due the position of the equilibrium
between 15 and 16 in the presence of Zn2+, which was
irreversibly sequestered on exposure of 12 to nitrilotriacetic acid.
8-(Benzoyloxy)-7-propenylquinoline (8). An oven-dried 250-mL
round-bottomed flask was placed under Ar and charged with 4 g (21.6
mol) of 7-propenyl-8-quinolinol 7 and 150 mL of anhydrous pyridine.
The solution was cooled at 0 °C, and to it was added 7.5 mL of benzoyl
chloride. The reaction mixture was allowed to stir at 0 °C for 5 min
and heated at 50 °C for 16 h. The reaction mixture was concentrated
under reduced pressure, and the residue was taken up in 100 mL of
ethyl ether. The organic phase was washed with 1 M HCl and then 2
M Na2CO3, dried over MgSO4, filtered, and concentrated to afford
6 g of 9 as a clear oil. Purification over silica (gradient of 10 to
40% EtOAc/hexanes) afforded a 3.15 g of a white solid (51%): 1H
NMR (500 MHz, CDCl3) δ 8.97 (d, J ) 4.3 Hz, 1H), 8.09 (d, J )
1.2 Hz, 2H), 8.05 (d, J ) 8.2 Hz, 1H), 7.72-7.30 (m, 6H), 6.77 (d,
J ) 15.9 Hz, 1H), 6.45 (dq, J ) 15.9, 6.6 Hz, 1H), 1.89 (d, J ) 6.6
Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 169.1, 150.3, 142.7, 141.3,
136.0, 133.3, 132.3, 130.6, 130.4, 130.3, 129.6, 129.4, 128.4, 128.2,
125.3, 123.9, 120.9, 26.7, 18.8; IR (KBr, cm-1) 3033, 1738, 1449, 1260,
1176, 1093; HRMS (CI, NH3) calcd for C19H15NO2 (M + 1) 290.1181,
found (M + 1) 290.1176, error 2 ppm; TLC Rf ) 0.30 (20% EtOAc/
hexanes).
7-Formyl-8-quinolinol (9). An oven-dried 100 mL round-bottomed
flask was charged with 1.9 g (6.57 mmol) of 8-(benzoyloxy)-7-
propenylquinoline (8), 120 mL of methanol, and 12 mL of dichlo-
romethane and cooled to -78 °C in a dry ice-2-propanol bath. A
stream of ozone was bubbled through the reaction for 30 min. TLC
was used to monitor the consumption of starting material. At -78
°C, Ar was bubbled through the bluish reaction mixture for 20 min to
remove excess ozone. Dimethyl sulfide (1.45 mL, 19.7 mmol) was
added, and the reaction mixture was allowed to warm to 25 °C and
stirred for 16 h. The reaction mixture was then concentrated under
reduced pressure. To the crude residue dissolved in 100 mL of ethanol
was added 0.99 g (7.23 mmol) of K2CO3 and 0.52 mL (32.8 mmol) of
Conclusions
We have therefore established that chemically and photo-
chemically reversible metal ion binding can be detected using
fluorescence spectroscopy with sensitivity for Zn2+ in the parts
per billion range (6.5 ppb for 11 in ethanol and 30 ppb for 15
(18) Bercovici, T.; Heiligman-Rim, R.; Fischer, E. Mol. Photochem. 1969,
1, 23-55. (b) Hirshberg, Y.; Knott, E. B.; Fischer, E. J. Chem. Soc. 1955,
3313-3321.
(19) Similar differences in metal ion-binding with Hg2+ were observed
in our earlier studies (ref 9).

Metal Ion Sensors with Parts per Billion SensitiVity
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3241
Figure 3. Fluorescence emission spectra of spiropyran 15 (10-5 M benzene, room temperature): (A) emission of “metal-free” solution; (B) emission
after addition of 1 equiv of ZnCl2; (C) emission after 30 s of irradiaion with visible light.
water. The mixture was heated at 70 °C for 2 h, cooled to room
temperaure, and then evaporated under reduced pressure. The residue
was taken up in 100 mL of chloroform. The organic phase was washed
with brine, dried over Na2SO4, filtered, and concentrated to afford 1 g
of 9 as a yellow oil. Crystallization from a methanol-dichloromethane
mixture afforded 608 mg (53%, two steps) of 7-formyl-8-quinolinol
Hanovia medium-pressure mercury lamp in a borosilicate glass im-
mersion well for 2 min and then the absorbance or emission spectrum
was immediately acquired (within 2 min). The sample was then
irradiated with a 150-W flood lamp, and the absorbance or emission
spectrum was again acquired.
Metal Ion Binding and Chemical Reversibility Experiments. A
10-5 M solution of the quinolinespiropyran 11 in ethanol was prepared.
A 3-mL aliquot of the solution was placed in a quartz absorbance or
fluorescence cuvette equipped with magnetic stirrer. The absorbance
spectrum was acquired or the sample was excited at 550 nm (the λmax
corresponding to the merocyanine form), and the emission spectrum
was acquired (average of three runs). Using a 10-µL syringe, the
appropriate volume of a 3 × 10-3 M solution of the metal chloride in
ethanol was added to the sample and the resulting solution was allowed
to stir in the dark. The absorbance or emission spectrum was acquired
(excitation at 572 nm) every 2 min until a maximum absorbance or
emission intensity was obtained (a maximum was reached immediately
in the case of absorbance and in ca. 1 min in the case of emission). A
0.3 M solution of nitrilotriacetic acid disodium salt in 10% EtOH/H2O
was prepared, and a 100-fold dilution was made to afford a 3 × 10-3
M solution of NTA in 10% EtOH/H2O. An appropriate volume of
this solution was added to the spiropyran-metal chelate solution using
a 10-µL syringe. The absorbance or emission spectrum (excitation at
550 nm) was then acquired. This binding-release cycle was repeated
as necessary to determine the extent of fatigue.
1
as an off-white solid: mp > 220 °C; H NMR (500 MHz, CDCl3) δ
10.36 (s, 1H), 8.94 (d, J ) 3.0 Hz, 1H), 8.15 (d, J ) 8.1 Hz, 1H), 7.74
(d, J ) 8.5 Hz, 1H), 7.57 (dd, J ) 7.8, 3.9 Hz, 1H), 7.35 (d, J ) 7.8
Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 192.9, 159.1, 149.4, 139.1,
136.1, 132.3, 125.8, 124.5, 118.4, 117.6; IR (KBr, cm-1) 3082, 2367,
2344, 1676, 1508, 1391, 1228; HRMS calcd for C10H7NO2 (M + 1)
173.0555, found (M + 1) 173.0548, error 4 ppm.
Spiropyran 11. An oven-dried 25-mL round-bottomed flask,
equipped with magnetic stirrer, was placed under Ar and charged with
200 mg (1.16 mmol) of 7-formyl-8-quinolinol (9) and 15 mL of
2-butanone. To this was added 0.185 mL (1.05 mmol) of 1,3,3-
trimethyl-2-methyleneindoline (10). The resulting reaction mixture was
heated at reflux for 16 h. After cooling to 25 °C, the reaction mixture
was concentrated under reduced pressure. The residue was purified
by flash chromatography over silica (1% to 10% MeOH/ CH2Cl2 with
1% Et3N) to afford 197 mg (57%) of 11 as a light purple solid: mp
193 °C (lit. mp 193 °C); 1H NMR (500 MHz, CDCl3) δ 8.48 (dd, J )
4.2, 1.7 Hz, 1H), 7.40 (dd, J ) 8.2, 1.5 Hz, 1H), 7.13 (dd, J ) 7.7, 1.0
Hz, 1H), 6.99 (dd, J ) 6.9, 0.9 Hz, 1H), 6.94 (s, 2H), 6.86(m, 1H),
6.60 (dd, J ) 8.2, 4.0 Hz, 1H), 6.56 (d, J ) 10.1 Hz, 1H), 6.41 (d, J
) 7.7 Hz, 1H), 5.51 (d, J ) 10.1 Hz, 1H), 2.62 (s, 3H), 1.47 (s, 3H),
1.11 (s, 3H); 13C NMR (125 MHz, C6D6) δ 149.2, 136.5, 135.9, 129.7,
129.2, 128.6, 127.8, 125.7, 125.3, 122.1, 121.7, 121.3, 119.9, 119.4,
118.7, 116.6, 107.0, 76.7, 64.1, 52.0, 28.9, 25.8, 24.4, 20.4; IR (neat,
cm-1) 2976, 2943, 2604, 2497, 1477, 1397, 1172, 1037; HRMS (CI,
NH3) calcd for C22H20N2O (M + 1) 329.1654, found (M + 1) 329.1650,
error 1.5 ppm.
Preparation of 15. Spiropyran 15. An oven-dried 50-mL round-
bottomed flask was placed under Ar charged with a magnetic stirring
bar, 58 mg (0.267 mmol) of 8-hydroxy-7-formyl-5-nitroquinoline (14),
20 mL of freshly distilled tetrahydrofuran, and 2.1 mL of DMSO. The
heterogeneous mixture was heated to 60 °C until dissolution was
achieved. To this yellow solution was added 57 µL (0.32 mmol) of
1,3,3-trimethyl-2-methyleneindoline (10), and the resulting mixture was
stirred under Ar at 25 °C for 16.5 h. The solvent was removed under
reduced pressure, and the residue was purified using flash chromatog-
raphy (pure CH2Cl2 and 1% Et3N, then gradient of MeOH in CH2Cl2
and 1% Et3N) affording 76 mg (77%) of the desired spiropyran 15 as
a gold-green solid: 1H NMR (500 MHz, C6D6) δ 8.91 (dd, J ) 8.8,
1.6 Hz, 1H), 8.37 (dd, J ) 4, 1.6 Hz, 1H), 8.01 (s, 1H), 7.25 (t, J )
7.5 Hz, 1H), 7.08 (d, J ) 7.5 Hz, 1H), 6.99 (t, J ) 7.5 Hz, 1H), 6.52
(d, J ) 7.5 Hz, 1H), 6.28 (d, J ) 10.2 Hz, 1H), 5.50 (d, J ) 10.2 Hz,
1H), 2.63 (s, 3H), 1.46 (s, 3H), 1.15 (s, 3H); 13C NMR (125 MHz,
C6D6) δ 155.8, 150.1, 147.9, 138.3, 137.1, 135.2, 131.8, 125.2, 124.3,
Metal Ion Binding Experiments Using 11. Photochromism at
Low Temperature. A 10-4 M solution of quinolinespiropyran 11 in
ethanol was prepared. A 0.5-mL aliquot of this solution was placed
in a quartz EPR tube (3 mm diameter). The Dewar-cuvette apparatus
was filled with a methanol/liquid N2 bath, and the EPR tube containing
the solution of spiropyran was cooled to -60 °C. The temperature of
the sample solution inside the EPR tube was monitored using a
temperature electrode. A blank was also prepared for use as a
background measurement. The sample was irradiated with a 450-W

3242 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Winkler et al.
124.1, 121.9, 120.6, 120.4, 114.2, 107.6, 107.5, 52.5, 28.7, 25.8, 20.2;
IR (neat, cm-1) 2917, 2849, 1609, 1574, 1514, 1470, 1390, 1274, 1223,
1110; TLC Rf ) 0.20 (10% MeOH/CH2Cl2 ); HRMS (CI/CH4) calcd
for C22H19N3O3 (M + H) 374.1504, found 374.1513, error 3 ppm.
Metal Ion Binding and Photohromism Experiments Using 15.
Photochromism at Low Temperature. A 10-4 M solution of
nitroquinolinespiropyran 15 in benzene was prepared and allowed to
equilibrate overnight in the absence of light at 25 °C. A 0.5-mL portion
of this solution was placed in a quartz EPR tube (3 mm diameter). The
Dewar-cuvette apparatus was filled with an ice-water bath, and the
EPR tube containing the solution of spiropyran was cooled to ca. 8
°C. A blank was also prepared for use as a background measurement.
The sample was irradiated with a 450-W Hanovia medium-pressure
mercury lamp in a borosilicate glass immersion well for 2 min, and
then the absorbance spectrum was immediately acquired (within 2 min).
The sample was then irradiated with a 150-W flood lamp, and the
absorbance spectrum was acquired.
10 µL of metal-free ethanol was added to the benzene solution of
nitroquinolinespiropyran. No significant change in the fluorescence
emission was observed. The absorbance or emission spectrum was
acquired (excitation at 550 nm) every 2 min until a maximum
absorbance or emission intensity was obtained (a maximum was reached
immediately in the case of absorbance and in ca. 4 min in the case of
emission). The sample was then irradiated with a 150-W flood lamp
for 30 s. Longer irradiation (up to 5 min) with visible light did not
have any effect on the extent of photoreversible metal ion binding in
any case. The absorbance or emission spectrum (excitation at 550 nm)
was immediately acquired. The sample was allowed to stir at 25 °C
in the dark, and the absorbance or emission spectrum was acquired
(excitation at 550 nm) every 2 min until a maximum absorbance or
emission intensity was obtained (again, this occurred immediately in
the case of absorbance and in ca. 4 min in the case of emission). This
cycle was repeated as necessary to determine the extent of photochromic
fatigue.
A 10-5 M solution of the nitroquinolinespiropyran 15 in benzene
was prepared and allowed to equilibrate overnight at 25 °C and in the
dark. A 3-mL aliquot of the solution was placed in a quartz absorbance
or fluorescence cuvette equipped with magnetic stirrer. The absorbance
spectrum was acquired or the sample was excited at 550 nm (the λmax
corresponding to the merocyanine form) and the emission spectrum
was acquired (average of three runs). Using a 10-µL syringe, the
appropriate volume of a ca. 10-3 M solution of the metal chloride in
ethanol was added to the sample and the resulting solution was allowed
to stir in the dark. A control experiment was also conducted in which
Acknowledgment. This paper is dedicated to Professor
Dieter Seebach on the occasion of his 60th birthday. The
generous financial support of the Office of Naval Research,
SmithKline Beecham, Wyeth-Ayerst, and Pfizer is gratefully
acknowledged. We also acknowledge Dr. Bin Shao for his
preliminary studies on the preparation of 11.
JA974181P