Optically switchable chelates: Optical control and sensing of metal ions

Article abstract of DOI:10.1021/jo7019898

(Chemical Equation Presented) This study introduces new concepts in the design, synthesis, and in vitro and in vivo characterization, manipulation, and imaging of organic chelates whose association with metal ions is rapidly and reversibly controlled by using light. Di- and tricarboxylic group bearing photochromes, nitrobenzospiropyran (nitroBIPS), undergo rapid and reversible, optically driven transitions between their spiro (SP) and fluorescent merocyanine (MC) states. The MC state of nitroBIPS-8-DA binds tightly to various metal ions resulting in specific shifts in absorption and fluorescence, and the dissociation constant for its Gadolinium complex in water is measured at ～5 μM. The metal-bound MC state is converted to the weaker-binding SP state with use of 543 nm light, while the SP to MC transition is complete with use of 365 or 720 nm (2-photon) light within several microseconds. Fluorescence imaging of the MC state of nitroBIPS-8-TriA was used to quantify the rate and efficiency of optical switching and to provide a real-time readout of the state of the optically switchable chelate within living cells.

Full text of DOI:10.1021/jo7019898

Optically Switchable Chelates: Optical Control and Sensing of
Metal Ions
Tomoyo Sakata,† David K. Jackson, Shu Mao, and Gerard Marriott*
Department of Physiology, UniVersity of Wisconsin, 1300 UniVersity AVenue, Madison, Wisconsin 53706
marriott@physiology.wisc.edu
ReceiVed September 20, 2007
This study introduces new concepts in the design, synthesis, and in vitro and in vivo characterization,
manipulation, and imaging of organic chelates whose association with metal ions is rapidly and reversibly
controlled by using light. Di- and tricarboxylic group bearing photochromes, nitrobenzospiropyran
(nitroBIPS), undergo rapid and reversible, optically driven transitions between their spiro (SP) and
fluorescent merocyanine (MC) states. The MC state of nitroBIPS-8-DA binds tightly to various metal
ions resulting in specific shifts in absorption and fluorescence, and the dissociation constant for its
Gadolinium complex in water is measured at ∼5 µM. The metal-bound MC state is converted to the
weaker-binding SP state with use of 543 nm light, while the SP to MC transition is complete with use
of 365 or 720 nm (2-photon) light within several microseconds. Fluorescence imaging of the MC state
of nitroBIPS-8-TriA was used to quantify the rate and efficiency of optical switching and to provide a
real-time readout of the state of the optically switchable chelate within living cells.
Spatio-temporal control of cell Ca2+ is a key feature of
signaling pathways that regulate transcription, proliferation, and
motility.1 Through the development and optimization of optical
probes for calcium2 it is now understood that spatio-temporal
control of Ca2+ serves as both an elemental molecular event in
cell signaling as well as a vehicle to transmit sensory events
throughout the cell.2-6 Further information on the role of
calcium transients in cell signaling has emerged from studies
using caged calcium ion probes, which undergo UV-driven
reactions that release Ca2+. For example, UV-irradiation of cell
loaded Nitr-5 and caged-EGTA can be used to generate spatially
defined transients of Ca2+ on a millisecond time scale.7-10
However, photochemical transitions using 2-nitrobenzyl groups
have complicated excited state chemistry11,12 with low quantum
yields (∼0.1).12 Cell based investigations with 2-nitrobenzyl
caged groups therefore require control studies to prove that any
response of a cell to UV-irradiation results from perturbation
of Ca2+ rather than an effect associated with UV light or the
photoproducts. Furthermore, the buildup of photoproducts and
their secondary reactions11 restricts the number of uncaging
events that can be performed within a single cell. We argue
that a more suitable probe to control divalent metal ions in cells
(7) Zacharias, D. A.; Baird, G. S.; Tsien, R. Y. Curr. Opin. Neurobiol.
2000, 10, 416-21.
* Address correspondence to this author.
†Current address: Genomics Institute of the Novartis Research Foundation,
10675 John Jay Hopkins Drive, San Diego, CA 92121.
(8) Adams, S. R.; Tsien, R. Y. Annu. ReV. Physiol. 1993, 55, 755-84.
(9) Adams, S. R.; Lev-Ram, V.; Tsien, R. Y. Chem. Biol. 1997, 4, 867-
78.
(1) Gomez, T. M.; Spitzer, N. C. J. Neurobiol. 2000, 44, 174-83.
(2) Palmer, A. E.; Tsien, R. Y. Nat. Protoc. 2006, 1, 1057-65.
(3) Tour, O.; Adams, S. R.; Kerr, R. A.; Meijer, R. M.; Sejnowski, T.
J.; Tsien, R. W.; Tsien, R. Y. Nat. Chem. Biol. 2007, 3, 423-31.
(4) Berridge, M. J. J. Physiol. 1997, 499 (Pt 2), 291-306.
(5) Marchant, J. S.; Parker, I. EMBO J. 2001, 20, 65-76.
(6) Torok, K.; Wilding, M.; Groigno, L.; Patel, R.; Whitaker, M. Curr.
Biol. 1998, 8, 692-9.
(10) Ellis-Davies, G. C.; Kaplan, J. H. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 187-91.
(11) Barth, A.; Martin, S. R.; Corrie, J. E. Photochem. Photobiol. Sci.
2006, 5, 107-15.
(12) Kantevari, S.; Hoang, C. J.; Ogrodnik, J.; Egger, M.; Niggli, E.;
Ellis-Davies, G. C. ChemBioChem 2006, 7, 174-80.
10.1021/jo7019898 CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/12/2007
J. Org. Chem. 2008, 73, 227-233
227

Sakata et al.
FIGURE 2. Absorption spectra of nitroBIPS-8-tert-butyl ester (2b)
(10 µM) in 1,2-propandiol after alternate irradiation with 365 nm light
(curves b and d) for 15 s and 546 nm light (curves a, c, and e) for
30 s.
SCHEME 2. Synthesis of NitroBIPS-8-triA (7) and Its AM
Ester (8)
FIGURE 1. Structures of di- and tricarboxyl-substituted photo-
chromes: dicarboxyl-bearing photochromes 2a (methyl ester), 2b (tert-
butyl ester), and 3 (free acid); tricarboxyl-bearing photochromes 6 (tert-
butyl ester), 7 (free acid), and 8 (acetoxymethyl (AM) ester).
SCHEME 1. Synthesis of NitroBIPS-8-DA (3)
described. For example, Kimura et al.21 showed that optical
transitions between the thermally stable SP and MC states of a
crowned spirobenzothiapyran could be controlled in acetonitrile
and that the MC state was stabilized by binding lithium ions.
All of these studies, however, were limited to investigations in
organic solvent and were complicated by thermal, metal ion,
and specific solvent effects on the equilibrium between SP and
MC states. Chelates for di- and trivalent metal ions that undergo
optically driven, rapid, and reversible SP-MC transitions in
aqueous solution and within living cells have not been reported.
Kimura’s group22 also demonstrated that the open state of
chromene crown ethers exhibited an improved affinity for
monovalent and specific alkali earth metal ions compared to
the closed state, but again these studies were limited to organic
solvent.
would undergo rapid and reversible, optically driven transitions
between two distinct states that exhibit marked differences in
their affinity for the metal ion. In addition, one of the two states
of the chelate should incorporate a fluorescence readout in order
(1) to determine the state of the switch at any time or location
in the cell, (2) to measure the free Ca2+ level, (3) to quantify
the rate and yield of the reaction, and (4) to assess effects of
adverse photochemistry such as photobleaching of the chelate.
Nitrobenzospiropyran (nitroBIPS) exhibits many of the ideal-
ized features outlined above including a well-documented ability
to undergo rapid and reversible, high quantum yield, optically
driven transitions between a colorless spiro (SP) state and a
colorful merocyanine (MC) state without the release of
photoproducts.13-16 Significantly, the MC state can also decay
from its excited state with the emission of red fluorescence.
Up to now several potential chelating nitroBIPS probes which
harbor polar chelating groups17 or crown ethers18-21 have been
On the basis of these considerations, we designed and
synthesized a new family of optical chelates for reversible
control of metal ion binding in vitro and in vivo. Chelating
groups were built into the nitroBIPS molecule on the 8-position
by using a synthetic approach described in Sakata et al.13 These
chelate-harboring nitroBIPS and their ester derivatives are
studied to demonstrate their potential for optical control of metal
ions in cells and for in vitro sensing of physiological relevant
(13) Sakata, T.; Yan, Y.; Marriott, G. J. Org. Chem. 2005, 70, 2009-
13.
(14) Sakata, T.; Yan, Y.; Marriott, G. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 4759-64.
(15) Inouye, M. Mol. Cryst. Liq. Cryst. Sci. Technol. 1994, 246, 169-
172.
(16) Kobatake, S.; Irie, M. In Photochromes. Annu. Rep. Prog. Chem.,
Sect. C 2003, 99, 277-313.
(20) Tanaka, M.; Ikeda, T.; Xu, Q.; Ando, H.; Shibutani, Y.; Nakamura,
M.; Sakamoto, H.; Yajima, S.; Kimura, K. J. Org. Chem. 2002, 67, 2223-
7.
(21) Tanaka, M.; Kamada, K.; Ando, H.; Kitagaki, T.; Shibutani, Y.;
Yajima, S.; Sakamoto, H.; Kimura, K. Chem. Commun. 1999, 1999, 1453-
1454.
(17) Deligeorgiev, T.; Minkovska, S.; Jejiazkova, B.; Rakovsky, S. Dyes
Pigm. 2002, 53, 101-108.
(18) Ahmed Saleh, A.; Tanaka, M.; Ando, H.; Iwamoto, H.; Kimura, K.
Eur. J. Org. Chem. 2003, 2437-2442.
(19) Salhin Abdussalam, M. A.; Tanaka, M.; Kamada, K.; Ando, H.;
Ikeda, T.; Shibutani, Y.; Yajima, S.; Nakamura, M.; Kimura, K. Eur. J.
Org. Chem. 2002, 655-662.
(22) Ahmed, S. A.; Tanaka, M.; Ando, H.; Iwamoto, H.; Kimura, K.
Eur. J. Org. Chem. 2003, 2437-2442.
228 J. Org. Chem., Vol. 73, No. 1, 2008

Optical Control and Sensing of Metal Ions
FIGURE 3. (A) Normalized absorption spectra of nitroBIPS-8-DA (3) in H2O at neutral pH in the presence of different metal ions (λmax): (i) Gd3+
(459 nm), Eu3+ (460 nm), Zn2+ (461 nm); (ii) Mn2+ (473 nm), Ni2+ (473 nm); (iii) Gd3+-EDTA (488 nm), Ca2+ (490 nm), no metal ion (492 nm).
(B) Titration of 3 with Gd3+. Normalized absorption spectra of 3 (6.9 µM) in H2O with Gd3+ at the ratio of Gd3+/3: no Gd3+, 0.01, 0.1, 0.2, 0.4,
0.8, 1, 2, 4, 8, and 10 (right to left). (C) Plot of the change in the absorption maximum wavelength of the GdCl3 complex. The data are fit to a single
binding site for Gd3+ with a dissociation constant of 5.2 µM. (D) Normalized, fluorescence emission spectra of 3 in H2O (6.9 µM) in the following
conditions: curve a, 3; curve b, 3 with GdCl3 (6.4 µM). The emission maxima occur at ∼600 and ∼550 nm for curves a and b, respectively.
metal ions such as Zn2+ 23 and Gd3+, the latter being used as a
contrast enhancing agent for MRI.24 Furthermore, the usefulness
of MC-fluorescence as a sensitive readout of the efficiency and
rate of optical switching in vitro is examined. These studies
also led to the new discovery that the SP state of the nitroBIPS
is efficiently excited with 2-photon light (700-730 nm) to form
the MC state. The nitroBIPS probes described in this report
represent our progress toward the design of optical switch
chelates that provide rapid and reversible optical control of
calcium and other physiologically relevant ions within living
cells.
groups at almost any site on the nitroBIPS scaffold. In this study
salicylaldehydes (1a, 1b, 5) prepared from commercially
available 3-chloromethyl-5-nitrosalicylaldehyde were coupled
with 1,3,3-trimethyl-2-methyleneindoline to yield the corre-
sponding esters (2a, 2b, 6).
Next, the ester groups were converted to carboxylic acids.
Because conventional saponification was unsuccessful possibly
due to the sensitivity of nitroBIPS to strong base, two different
hydrolytic reactions were used to generate the free carboxylic
acid forms (3, 7): the first is bis(tributyltin)oxide-mediated
hydrolysis of the methyl ester (2a), and the second is based on
trifluoroacetic acid (TFA)-mediated hydrolysis of the tert-butyl
esters (2b, 6). The obtained chelates 3 and 7 were subjected to
Chelex column to remove possible trace amounts of metal cation
prior to spectroscopic studies. NitroBIPS-triA (7) was further
converted to acetoxymethyl ester (AM ester) (8) by coupling
with bromomethyl actate.
Results and Discussion
Design and Synthesis of Optically Switchable Chelates.
The metal ion binding function was built into the nitroBIPS
photochrome by introducing a di- or tri-carboxylate-bearing side
group at the 8-position (Figure 1 compounds 3 and 7 respec-
tively). First, ester-bearing nitroBIPS (2a, 2b, 6) were synthe-
sized based on the published methods,13 as summarized in
Schemes 1 and 2 and detailed in the Supporting Information.
Combinatorial in design, the syntheses involved a coupling
reaction of indoline derivatives with ester-bearing salicylalde-
hydes. This approach allows for the introduction of carboxyl
Spectroscopic and Optical Switching Properties of Nitro-
BIPS-8-tert-butyl Ester (2b). The absorption spectra of the SP
state (SP absorption spectra) of tert-butyl ester (2b) in 1,2-
propandiol has the maximum value at 340 nm and the half-
maximal at 375 nm (Figure 2, curve a) and is largely insensitive
to solvent polarity whereas the absorption spectrum of the highly
polar MC state of 2b is sensitive to general and specific solvent
effects (data not shown), which is consistent with our previous
results.13 Optically driven transitions between the SP and MC
states of 2b occur with high efficiency in organic solvents as
(23) Burdette, S. C.; Walkup, G. K.; Spingler, B.; Tsien, R. Y.; Lippard,
S. J. J. Am. Chem. Soc. 2001, 123, 7831-41.
(24) Cohen, S. M.; Xu, J.; Radkov, E.; Raymond, K. N.; Botta, M.; Barge,
A.; Aime, S. Inorg. Chem. 2000, 39, 5747-56.
J. Org. Chem, Vol. 73, No. 1, 2008 229

Sakata et al.
FIGURE 4. (A) Time course of the thermal transition (SP to MC) of nitroBIPS-8-DA (3) (4 µM) in the absence of GdCl3 in water (λmax at 491
nm at 14 h). Each spectrum was recorded at the following time points: 15 min; 30 min; 1 h; 3 h; and 14 h. (B) Time course of the thermal transition
(SP to MC) of 3 (4 µM) in the presence of 6.4 µM of GdCl3 (λmax at 459 nm at 14 h) in H2O. Each spectrum was recorded at the following time
points: 15 min; 30 min; 1 h; 3 h; and 14 h. (C) Optical switching between the SP and MC states of 3 (4 µM) with the presence of GdCl3 (6.4 µM)
in H2O. Alternate irradiation was performed with 365 nm light (curves b and d) for 15 s and 546 nm light (curves a, c, and e) for 1 min. (D) Optical
switching between the SP and MC states of 3 (4 µM) in 1,2-propandiol/Tris, pH 7 (99:1) at 20 °C. Alternate irradiation was performed with 365
nm light (curves b and d) for 15 s and 546 nm light (curves a, c, and e) for 30 s.
shown in Figure 2; the SP to MC transition for a 10 µM solution
is complete within a 15 s irradiation of 365 nm light (UV),
while the MC state is converted to the SP state following 30 s
of irradiation with 546 nm light (VIS). The SP and MC states
can be interconverted over many UV-vis irradiation cycles
without significant changes in the absorption spectrum or
evidence of fatigue (Figure 2).
Metal Ion Chelating Properties of NitroBIPS-8-DA (3) in
Water. Conformational change of nitroBIPS and the effect of
chelation on its electron density distribution result in the shift
on the compound’s absorption spectrum. Figure 3A shows that
di- and trivalent metal ions bind to the MC state of nitroBIPS-
8-DA (3) over a µM concentration range. The value of the
maximum MC-absorption wavelength was shifted from 492 nm
for free acid 3 (i.e., without metal ions) to 459 nm with the
presence of 100 µM Gd3+ in water, and the range of this spectral
shift was specific for each chelated metal ion.
Gadolinium Ion Complexes of NitroBIPS-8-DA (3) in
Water. Metyl ester 2a does not exhibit any significant affinity
for Gd3+, which caused the largest shift of nitroBIPS-8-DA (3),
or divalent metal ions, nor do other nitroBIPS lacking a carboxyl
group described by Sakata et al.13 (data not shown). Also, Gd3+
does not show any sensible binding to the SP state of nitroBIPS-
8-DA (3) in water up to a concentration of 1 mM as might be
detected by monitoring changes in the SP-absorption spectrum
(data not shown). On the other hand, the MC state of 3 was
shown to bind to a single Gd3+ ion with a dissociation constant
of 5.2 µM in water (Figure 3B,C). Interestingly, the MC state
of 3 also exhibits a fluorescence emission whose energy and
quantum yield is metal ion specific, as shown for the effect of
Gd3+ in Figure 3D (curve a, Gd3+ free; curve b, with the
presence of Gd3+).
NitroBIPS-8-DA (3) and related chelates having one or more
carboxyl groups in close proximity to the spiro bond exhibit a
pronounced reverse photochromism; they undergo a SP to MC
transition even in the dark (Figure 4A,B). The origin of reverse
photochromism is not fully understood, although nitroBIPS
lacking a carboxyl group also show his behavior in acidic
medium.25 Our results suggest that the built-in carboxyl group
might accelerate the SP to MC transition by stabilizing
protonation of the generated oxy-anion. The rate of reverse
photochromism for nitroBIPS-8-DA (3) is not significantly
affected by the presence of Gd3+ over the initial phase of the
measurement period (Figure 4A,B).
Absorption studies show that the UV-driven SP to MC
transition of nitroBIPS-8-DA (3) does occur in water but with
low efficiency (Figure 4C). Thus, the SP state of 3 in the
presence of chelated Gd3+ (Figure 4C, curve a) led to a slight
increase in the intensity of the MC-absorption when irradiated
for 1 min with 365 nm light (Figure 4C, curve b). By considering
the large increase of absorption observed during the thermal
transition (Figure 4A,B), this result suggested that the UV-driven
SP to MC transition was incomplete. However, the UV-driven
(25) Raymo, F. M.; Giordani, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
4941-4944.
230 J. Org. Chem., Vol. 73, No. 1, 2008

Optical Control and Sensing of Metal Ions
MC state can be brought back to the SP state by using 543 nm
irradiation, and additional UV-irradiation of the SP state (Figure
4C, curve c) followed by visible light excitation showed that
transitions between the SP state (Figure 4C, curves a, c, and e)
and the MC-state (Figure 4C, curves b and d) are reversible.
Optical and Thermo-optical Manipulation of NitroBIPS-
8-DA in Organic Solvents. In contrast to studies performed in
water, nitroBIPS-8-DA (3) dissolved in 1,2-propandiol/water
(99:1) at pH 7.0 undergoes robust, high fidelity, 365 and 546
nm-driven transitions between the SP and MC states respectively
in the presence (Figure 4D, curves a-e) or absence of Gd3+
(data not shown). A comparison of the results shown in parts
C and D of Figure 4 suggests that water plays a strong role in
decreasing the UV-driven quantum yield for the SP to MC
transition and promoting reverse photochromism in nitroBIPS-
8-DA. The low quantum yield for the UV-driven SP to MC
reaction is not unexpected since the reaction is greatly influenced
by solvent polarity even for non-carboxyl-bearing nitroBIPS.26
On the other hand the strong reverse photochromism found for
carboxyl-bearing nitroBIPS switches in water suggests that
carboxyl groups play either a direct or an indirect role in
stabilizing the oxy-anion of the MC state. This complicating
factor might be significantly reduced by locating the carboxyl
groups away from this region of the nitroBIPS molecule.
Optical Manipulation of Chelates within Living Cells. The
AM-ester (acetoxymethyl ester, 8) of nitroBIPS-8-TriA (7) and
ethyl esters of other chelate bearing nitroBIPS are capable of
diffusing across the plasma membrane of living cells. On the
basis of other studies with AM-esters of fluorescent calcium
indicators,8 the AM-ester groups of 8 are presumably hydrolyzed
by intracellular esterases to generate the membrane impermeable
carboxylate 7. For example, live NIH 3T3 cells treated with 10
µM of the AM-ester (8) in growth medium for 30 min exhibited
a strong and uniform MC-fluorescence in cells upon excitation
with 535 nm light (Figure 5A). Similar results were obtained
for the ethyl ester of a tetracarboxyl-substituted nitroBIPS (data
not shown). Support for the claim that AM and ethyl esters of
optical switch chelates are hydrolyzed by intracellular esterases
was obtained from a separate study using the methyl ester 2a,
which rapidly traverses the plasma membrane but is not a
substrate for esterases, and so the intracellular MC-fluorescence
of 2a rapidly disappears from the cell upon washing with fresh
medium (data not shown).
Optical switching of nitroBIPS-8-TriA (7) within living NIH
3T3 cells was demonstrated by using a 2-photon fluorescence
microscope. Irradiation of cells with 543 nm light was used to
image the distribution of the MC form of the chelate in cells
and to quantify optical switching between the SP and MC states.
Thus, quantifying the change in red MC-fluorescence during
optical switching allowed us to show that several 543 nm scans
of the cell decreased the MC-fluorescence to a baseline value
as a result of the MC to SP transition (see montage in Figure
5A). The SP to MC transition was triggered within the same
cell by scanning the field with 720 nm light, which led to a
2-photon-mediated excitation of the SP state and photochemistry
to the MC state. The laser power of the 720 and 535 nm lasers
used in these experiments was low and so several scans were
required for complete interconversion of the SP and MC states.
On the other hand, other studies using higher 2-photon excitation
power showed that a single scan was sufficient to quantitatively
convert SP to MC within the focal plane (data not shown).
FIGURE 5. (A) A montage of MC-fluorescence images of NIH 3T3
cells loaded with the AM-ester (8) of optical switch chelate obtained
by confocal imaging using an excitation wavelength of 535 nm and
emission >590 nm. The chelate is retained in cells after several
washings with culture medium. The MC state was generated by serial
scannings of the image field with 720 nm (2-photon) light. (B) The
intensity of MC fluorescence in a selected area of the image was used
to show the reversibility of transitions between the two states of
nitroBIPS-8-TriA (SP to MC, 720 nm, blue circles; MC to SP, 535
nm, red circles). The uniform distribution of the MC fluorescence
suggests that the nitroBIPS-8-TriA (7) is contained within the cyto-
plasm. Rapid and reversible, high-fidelity transitions between the SP
and MC states are shown for 10 cycles.
Imaging and analysis of the intracellular MC fluorescence
of chelate 7 as a function of 2-photon and 543 nm irradiation
shows that the chelate undergoes numerous transitions between
the SP and MC states in response to alternating irradiation with
720 and 543 nm light (Figure 5A,B). The fastest 720 nm,
2-photon scan possible in our system showed that the SP to
MC reaction was complete within the 6 µs pixel dwell time of
the laser (data not shown). The gradual increase in MC-
fluorescence evidenced prior to 2-photon excitation of each
irradiation cycle (Figure 5B) suggests that the power of the 543
nm laser used in this study was too low to convert the entire
MC population to SP. The data shown in Figure 5B also show
that the optically switchable chelate (7) exhibits little fatigue
over the 30 cycles of alternate 2-photon/1-photon scanning.
The results shown in this study suggest that nitroBIPS-8-
TriA (7) unexpectedly undergoes normal photochromism in cells
and, unlike nitroBIPS-8-DA in water, it exhibits a robust
2-photon-driven transition between the SP and MC states. It is
long known that anionic, aromatic dyes such as fluorescent
calcium ion indicators, i.e., Fura-2, bind to cell proteins, and in
some cases this leads to a change in the functional properties.
Interestingly, the quantum yield for the SP to MC transition of
the cell permeable optical switch chelates used in this study is
improved in living cells. We speculate that chelate 7 engages
in similar nonspecific interactions with cell proteins overcoming
reverse photochromism, presumably by shielding the probe from
bulk water.
(26) Go¨rner, H. Phys. Chem. Chem. Phys. 2001, 3, 416-423.
J. Org. Chem, Vol. 73, No. 1, 2008 231

Sakata et al.
(s, 4H), 4.07 (s, 2H), 8.22 (d, J ) 3.8 Hz, 1H), 8.65 (d, J ) 3.8
Hz, 1H), 10.44 (s, 1H).
Conclusion
This study introduced new concepts and principles in the
design of optically switchable chelates for rapid and reversible
1- and 2-photon mediated control of metal ions in solution and
within living cells. By incorporating the iminodiacetate group
into the photochromic nitroBIPS scaffold, we have developed
chelates that can take two structurally different states, SP and
MC. The synthesis involves a coupling reaction of indoline and
ester-bearing salicylaldehyde followed by hydrolysis of the ester
groups, and this approach can easily be adapted to incorporate
a variety of chelating groups on different positions on the BIPS
scaffold. The ability to change the relative positions of the
chelating carboxyl groups and generated oxyanion between the
SP and MC states provides a simple and powerful approach to
optically modulate the metal ion binding affinity of the
nitroBIPS probe. In vitro studies showed that the MC state of
nitroBIPS-8-DA was an effective chelator of Gd3+, and other
metal ions that may be distinguished on the basis of the
absorption and emission properties of each respective complex.
The nitroBIPS harboring ethyl and acetoxymethy esters were
shown to cross the cell membrane and were retained following
esterase-mediated hydrolysis. Optical switching between the SP
and MC states of the chelate and associated modulation of metal
ion affinity was demonstrated in vitro and in living cells by
using 1- and 2-photon excitation for the SP to MC transition
and 543 nm for the MC to SP transition. Given the relatively
low affinity of the MC state of the nitroBIPS-8-DA chelatefor
Ca2+, we did not expect to see any Ca2+ related response of
the cell during optical control of the SP and MC states of the
related chelate 7. However, these studies clearly demonstrate
the feasibility of controlling and imaging cell Ca2+ by using
probes related to chelate 7 whose MC state exhibits a higher
General Synthetic Method of NitroBIPS (2a, 2b, 6). A THF
solution (2 mL) of salicylaldehyde 1a (75 mg, 0.22 mmol) and
1,3,3-trimethyl-2-methyleneindoline (51 mg, 0.29 mmol) was stirred
at rt for 2 h. Following the addition of saturated Na2CO3 the product
was extracted with CH2Cl2 and dried over Na2SO4 and after
evaporating solvent, the residue was subjected to column chroma-
tography (SiO2; eluent, hexane:AcOEt ) 5:1) to afford 2a (51 mg,
35% based on indoline).
NitroBIPS-8-dimethyl Ester (2a). Yield 35% based on indoline;
MS (EI) 495 (M+, 5), 422 (2), 84 (100); HRMS (EI) M+495.1998
1
(calcd 495.2006); H NMR (CDCl3) δ 1.20 (s, 3H), 1.27 (s, 3H),
2.69 (s, 3H), 3.27 (s, 4H), 3.60 (s, 3H), 3.59 (d, J ) 14.2 Hz, 1H),
3.66 (d, J ) 14.2 Hz, 1H), 5.87 (d, J ) 10.1 Hz, 1H), 6.54 (d, J )
7.6 Hz, 1H), 6.86 (dd, J ) 7.6, 7.6 Hz, 1H), 6.92 (d, J ) 10.1 Hz,
1H), 7.07 (d, J ) 7.6 Hz, 1H), 7.17 (dd, J ) 7.6, 7.6 Hz, 1H), 7.94
(d, J ) 2.6 Hz, 1H), 8.12 (d, J ) 2.6 Hz, 1H).
NitroBIPS-8-di-tert-butyl Ester (2b). Yield 63% based on
indoline; MS (EI) 579 (M+, 30), 478 (48), 464 (61), 408 (60), 336
(69), 335 (69), 159 (64), 83 (100); HRMS (EI) M+579.2948 (calcd
579.2945); 1H NMR (CDCl3) δ 1.19 (s, 3H), 1.27 (s, 3H), 1.38 (s,
18H), 2.70 (s, 3H), 3.23 (s, 4H), 3.61 (s, 2H), 5.85 (d, J ) 10.1
Hz, 1H), 6.53 (d, J ) 7.4 Hz, 1H), 6.84 (ddd, J ) 1.0, 7.4, 7.4 Hz,
1H), 6.92 (d, J ) 10.1 Hz, 1H), 7.06 (dd, J ) 1.0, 7.4 Hz, 1H),
7.15 (ddd, J ) 1.2, 7.4, 7.4 Hz, 1H), 7.92 (d, J ) 2.6 Hz, 1H),
8.25 (d, J ) 2.6 Hz, 1H).
NitroBIPS-8-tri-tert-butyl Ester (6). Yield 30% based on 4;
HRMS (ESI) [M + H]+ 737.7140 (calcd 737.4125); 1H NMR
(CDCl3) δ 1.20 (s, 3H), 1.28 (s, 3H), 1.38 (s, 9H), 1.43 (s, 18H),
2.66 (m, 4H), 2.70 (s, 3H), 3.10 (s, 2H), 3.35 (s, 4H), 3.56 (s, 2H),
5.86 (d, J ) 10.2 Hz, 1H), 6.54 (d, J ) 7.5 Hz, 1H), 6.87(ddd, J
) 1.3, 7.5, 7.5 Hz, 1H), 6.93(d, J ) 10.2 Hz, 1H), 7.07(dd, J )
0.6, 7.5 Hz, 1H), 7.18 (ddd, J ) 1.3, 7.5, 7.5 Hz, 1H), 7.92 (d, J
) 3.0 Hz, 1H), 8.22 (d, J ) 3.0 Hz, 1H).
NitroBIPS-8-DA (3). From 2a: To a benzene solution (0.5 mL)
of 2a (16 mg, 32 µmol) was added Bu3SnO (72 mg, 120 µmol),
and the reaction mixture was refluxed for 1.5 days. After 10 min
of stirring with 0.5 N HCl, the product was extracted with EtOAc,
concentrated, and subjected to column chromatography (Sephadex
LH-20; eluent, hexane:CH2Cl2:MeOH ) 2:1:1) to give an impure
fraction containing the desired compound. Staring reagents were
removed by extraction with CH2Cl2, and the aqueous layer was
basified and extracted with CH2Cl2 to afford 3 (3 mg, 20%). From
2b: To a CH2Cl2 solution (0.5 mL) of 1b (40 mg, 69 µmol) was
added TFA (0.5 mL), and the reaction mixture was stirred at rt for
3.5 h. After evaporation of the solvent, the residue was subjected
to column chromatography (Sephadex LH-20; eluent, hexane: CH2-
Cl2:MeOH ) 2:1:1) to afford a red oil 3 (35 mg, 80%). MS
affinity for Ca2+
.
To the best of our knowledge this is the first description of
2-photon excitation-mediated SP to MC transition in nitroBIPS
related switches. This property opens up the opportunity to
control the two states of nitroBIPS related optical switch by
using only 2-photon excitation, i.e., 720 nm for the SP to MC
transition and 980 nm for the MC to SP transition. This feature
should prove useful in studies that require control of metal ions
within tissue slices and under the skin of animals.
Experimental Section
1
(MALDI) 338 (335 + Na), 335 [M - N(CH2CO2H)2]; H NMR
Synthesis. All reagents and starting materials except for triester
4 are commercially available. 4 was synthesized according to
Achifelu et al.27
General Synthetic Method of Salicylaldehyde (1a, 1b, 5). To
a THF solution (8 mL) of di-tert-butyl iminoacetate (504 mg, 2.1
mmol) and Et3N (540 µL, 3.9 mmol) was added 3-(chloromethyl)-
5-nitrosalicylaldehyde (435 mg, 2.0 mmol). After refluxing for 4
h, the reaction mixture was filtered and concentrated to give a
yellow oil of 1b as a mixture with Et3N (1b:Et3N ) 10:7), which
was used for the subsequent reaction without further purification.
(acetone-d6) δ 1.25(br s, 3H), 1.36 (br s, 3H), 2.76 (s, 3H), 3.42 (s,
4H), 3.74 (s, 2H), 6.05 (d, J ) 10.1 Hz, 1H), 6.63 (d, J ) 7.4 Hz,
1H), 6.85 (dd, J ) 7.4, 7.4 Hz, 1H), 7.15 (d, J ) 7.4 Hz, 1H), 7.16
(dd, J ) 7.4, 7.4 Hz, 1H), 7.15 (dd, J ) 7.4, 7.4 Hz, 1H), 8.09 (d,
J ) 2.9 Hz, 1H), 8.27 (d, J ) 2.9 Hz, 1H).
NitroBIPS-8-TriAM (8). To a CH2Cl2 solution (0.5 mL) of tert-
butyl ester 6 (10 mg, 15 µmol) was added TFA (0.5 mL), which
was then stirred at rt for 5 h. After evaporation of the solvent, the
residue was subjected to column chromatography (Sephadex LH-
20; eluent, hexane: CH2Cl2:MeOH ) 2:1:1) to afford a red oil
containing nitroBIPS-8-TriA (7), which was then dissolved in CH3-
CN (1 mL). To this solution were added bromomethylacetate (14
mg, 92 umol) and Et3N (20 µL, 144 umol) and the mixture was
stirred over night. After concentration, the residue was purified on
preparative TLC (hexane:EtOAc ) 1:1) to afford 8 (2.4 mg, 20%
based on 6). HRMS (ESI) [M + H]+ 785.2890 (calcd 785.2881);
1H NMR (CDCl3) δ 1.20 (s, 3H), 1.28 (s, 3H), 2.12 (s, 6H), 2.18
(s, 3H), 2.64 (s, 4H), 2.70 (s, 3H), 3.20 (s, 2H), 3.50 (s, 4H), 3.57
(m, 2H), 5.67 (s, 2H), 5.73 (s, 4H), 5.88 (d, J ) 10.2 Hz, 1H),
3-[N,N-Bis(methyloxycarbonylmethyl)aminomethyl]-5-nitro-
1
salicylaldehyde (1a). H NMR (CDCl3) δ 3.62 (s, 4H), 3.78 (s,
6H), 4.12 (s, 2H), 8.35 (d, J ) 2.7 Hz, 1H), 8.61 (d, J ) 2.7 Hz,
1H), 10.36 (s, 1H).
3-[N,N-Bis(tert-butyloxycarbonylmethyl)aminomethyl]-5-ni-
trosalicylaldehyde (1b). H NMR (CDCl3) δ 1.50 (s, 18H), 3.46
1
(27) Achilefu, S.; Wilhelm, R. R.; Jimenez, H. N.; Schmidt, M. A.;
Srinivasan, A. J. Org. Chem. 2000, 65, 1562-5.
232 J. Org. Chem., Vol. 73, No. 1, 2008

Optical Control and Sensing of Metal Ions
6.54 (d, J ) 7.4 Hz, 1H), 6.86 (ddd, J ) 1.2, 7.4, 7.4 Hz, 1H),
6.94 (d, J ) 10.2 Hz, 1H), 7.07 (dd, J ) 0.5, 7.4 Hz, 1H), 7.18
(ddd, J ) 1.2, 7.4, 7.4 Hz, 1H), 7.94 (d, J ) 2.8 Hz, 1H), 8.09 (d,
J ) 2.8 Hz, 1H).
Light-Directed Switching. Switching of the probes described
in this work was achieved by irradiating the sample (120-1000
µL) with the 365 nm line of a hand-held UV lamp or the 546 nm
line of a 100 W Hg-arc lamp.
Microscopy. Single- and two-photon imaging was performed
on an LSM510 microscope with a ×40 1.2NA Objective and
incorporating a mode-locked titanium:sapphire laser oscillator.
Multiphoton photoactivation of the SP state of the optical switch
to the MC state was achieved by using 720 nm light. Excitation of
the MC state, which leads to its conversion to the SP state or return
to the MC ground state with emission of a red photon, was achieved
by using 543 nm He-Ne laser excitation. MC-fluorescence was
collected through a 565-615 nm band-pass filter with an infrared
blocking property.
for 30 min and washed 3-4 times with fresh medium. Loaded cells
were scanned sequentially with one switching cycle consisting of
1-5 scans at 720 nm (2-photon) followed by 5-10 scans at 543
nm. The optical switching cycle was repeated 10-30 times for each
experiment. Fluorescence of the MC state (and conversion of the
MC to SP state) in loaded cells was achieved by scanning a 512 ×
512-field with 543 nm light within a second. The SP to MC
transition was usually achieved by scanning the field at the same
rate with 2-photon light. The optimal excitation wavelength for the
SP to MC transition was 715 nm.
Acknowledgment. This work was supported by a grant
(5R01EB005217-03) awarded to G.M. We thank Drs. David
Piston and Richard Benninger for use of their 2-photon confocal
fluorescence microscope.
Supporting Information Available: 1H NMR spectra of the
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Cell Studies. Ester derivatives of nitroBIPS-TriA in DMSO were
added to the cell medium (DMEM/10% FCS) to achieve a final
concentration of ∼10 µM solution. Cells were incubated at 37 °C
JO7019898
J. Org. Chem, Vol. 73, No. 1, 2008 233