Photoswitchable Fluorescent Dyads Incorporating BODIPY and [1,3

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Photoswitchable Fluorescent Dyads Incorporating BODIPY and [1,3]Oxazine Components Erhan Deniz,† Shuvasree Ray,† Massimiliano Tomasulo,† Stefania Impellizzeri,† Salvatore Sortino,*,‡ and Franc¸isco M. Raymo*,† Department of Chemistry, UniVersity of Miami, 1301 Memorial DriVe, Coral Gables, Florida, 33146-0431, United States and Dipartimento di Scienze Chimiche, UniVersita` di Catania, Viale Andrea Doria 8, Catania, I-95125, Italy ReceiVed: July 29, 2010; ReVised Manuscript ReceiVed: September 15, 2010

We designed and synthesized three compounds incorporating a BODIPY fluorophore and an oxazine photochrome within the same molecular skeleton and differing in the nature of the linker bridging the two functional components. The [1,3]oxazine ring of the photochrome opens in less than 6 ns upon laser excitation in two of the three fluorophore-photochrome dyads. This process generates a 3H-indolium cation with a quantum yield of 0.02-0.05. The photogenerated isomer has a lifetime of 1-3 µs and reverts to the original species with first-order kinetics. Both photochromic systems tolerate hundreds of switching cycles with no sign of degradation. The visible excitation of the dyads is accompanied by the characteristic fluorescence of the BODIPY component. However, the cationic fragment of their photogenerated isomers can accept an electron or energy from the excited fluorophore. As a result, the photoinduced transformation of the photochromic component within each dyad results in the effective quenching of the BODIPY emission. Indeed, the fluorescence of these photoswitchable compounds can be modulated on a microsecond time scale with excellent fatigue resistance under optical control. Thus, our operating principles and choice of functional components can ultimately lead to the development of valuable photoswitchable fluorescent probes for the super-resolution imaging of biological samples. Introduction 1

Fluorescence microscopy, in combination with appropriate labeling methodologies,2 offers the opportunity to image biological samples noninvasively in real time. As a result, this convenient imaging technique has become an essential analytical tool in the biomedical laboratory. Nonetheless, the phenomenon of diffraction3 imposes stringent limitations on the resolving power of conventional fluorescence microscopes. Specifically, fluorescence objects separated by less than 0.5 µm along the optical axis or 0.2 µm in the focal plane cannot be distinguished.1 These values are significantly greater than the physical dimensions of biomolecules. Thus, the subtle factors regulating biological processes and structures at the molecular level cannot be appreciated with this technique. Valuable strategies to overcome the diffraction barrier and improve the axial and lateral resolutions of conventional microscopes are starting to emerge.4-11 These methods are based on the patterned illumination of the specimen or the reiterative identification of individual fluorophores. For example, the patterned irradiation of a biological sample labeled with photoswitchable fluorophores offers the opportunity to record images with nanoscaled resolution. This protocol, however, requires the use of probes able to undergo reversible saturable optically linear fluorescence transitions (RESOLFT). Specifically, the probes must be designed to emit upon excitation at one wavelength (λON) and switch to a nonemissive state when excited at another wavelength (λOFF). Under these conditions, the illumination of the sample with pairs of beams at λON and * To whom correspondence should be addressed. E-mail: [email protected]; [email protected]. † University of Miami. ‡ Universita` di Catania.

λOFF, producing overlapped circular and doughnut-shaped spots, respectively, on the focal plane, can be exploited to localize the emissive species in nanoscaled regions and record images with subdiffraction resolution. Stimulated emission depletion (STED) and intersystem crossing (ISC) have been exploited to switch probes from emissive to nonemissive states.4 However, the high irradiation intensity required for STED and the reactivity of the triplet states populated after ISC complicate the application of these processes to super-resolution imaging. In principle, these limitations can be overcome with the design of RESOLFT based on the photoinduced interconversions of photochromic compounds.12-17 Indeed, these molecules switch reversibly between states with distinct absorption spectra under the influence of optical stimulations and can be exploited to photomodulate the emission of complementary fluorophores.17-19 Indeed, their use as fluorescent labels for RESOLFT imaging has already been explored with prototypical examples.20 In the wake of these promising results, we designed the fluorophore-photochrome dyad 1a (Figure 1).21 This molecule incorporates a fluorescent boron dipyrromethene (BODIPY) component, which emits at 540 nm upon irradiation at 480 nm (λON), and a photochromic spiropyran component, which switches to the corresponding merocyanine upon irradiation at 254 nm (λOFF). The photoinduced transformation of 1a into 1b activates electron and energy transfer pathways, which culminate in the effective quenching of the BODIPY fluorescence. The photogenerated species 1b has a lifetime of 270 s in acetonitrile at 25 °C and eventually reverts to 1a with first-order kinetics. Thus, the fluorescence of this particular system can be switched off and on simply by turning on and off, respectively, an ultraviolet source. However, the relatively slow reisomerization kinetics and poor fatigue resistance of this system are not compatible with RESOLFT

10.1021/jp107116d  2010 American Chemical Society Published on Web 10/13/2010

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Figure 1. Photoinduced and reversible transformation of 1a into 1b.

imaging. Indeed, a full switching cycle from 1a to 1b and back can only be completed on a time scale of several minutes, and 25% of the original fluorescence is lost after only five cycles. Thus, the fatigue resistances and switching speeds of this fluorophore-photochrome dyad must be improved significantly in view of possible applications in superresolution imaging, which require microsecond switching times and the ability to tolerate tens of switching cycles. In this article, we report the synthesis of three novel dyads with improved performance and characterization of their photochemical and photophysical properties with a combination of steady-state and time-resolved spectroscopic measurements. Results and Discussion Design and Synthesis. The [1,3]oxazine ring of 2a (Figure 2) opens in less than 6 ns to generate 2b with a quantum yield of 0.07 in acetonitrile at 20 °C upon ultraviolet irradiation.22 The photogenerated isomer 2b has a lifetime of 2 µs under these conditions and eventually reverts to 2a with first-order kinetics. Thus, a full switching cycle can be completed on a microsecond time scale with this photochromic system. Furthermore, this species survives unaffected hundreds of switching cycles even in the presence of molecular oxygen. Thus, the introduction of this photochromic oxazine within 1a in place of the spiropyran component should translate into the generation of fluorophorephotochrome dyads with switching speeds and fatigue resistances compatible with RESOLFT imaging. On the basis of these considerations, we designed the BODIPY-oxazine dyads 3a-5a and synthesized these compounds in 3-8 steps starting from commercial and known precursors. We prepared the fluorophore-photochrome dyad 3a in five steps, starting from 4-methoxyphenyl hydrazine (Figure 3).23 In particular, we condensed this precursor with 3-methyl-2butanone, in the presence of p-toluene sulfonic acid (PTSA), to generate the 3H-indole 6. Then, we condensed 6 with 4-dimethylamino benzaldehyde, in the presence of hydrogen bromide and trifluoroacetic acid (TFA), to produce the extended 3H-indole 7. After cleavage of the methoxy group with boron tribromide, we reacted the resulting compound 8 with 9, under the assistance of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), to produce 10. Finally, we condensed 10 with 2-chloromethyl-4-nitrophenol to yield the target molecule 3a. We synthesized the fluorophore-photochrome dyad 4a in three steps, starting from 2,3,3-trimethyl-3H-indole (Figure 4). Specifically, we condensed this precursor with N-methyl-N-(2hydroxyethyl)-4-aminobenzaldehyde, in the presence of hydrogen bromide, to generate the extended 3H-indole 11. Then, we reacted 11 with 9, under the assistance of DCC and DMAP, to

Deniz et al. produce 12 and condensed this compound with 2-chloromethyl4-nitrophenol to yield the target molecule 4a. We prepared the fluorophore-photochrome dyad 5a in three steps, starting from 2,3,3-trimethyl-3H-indole (Figure 5). In particular, we alkylated 4-hydroxy benzaldehyde with 1,2dibromoethane, in the presence of potassium carbonate, and reacted the resulting bromide 13 with sodium azide to produce 14. Then, we treated 14 with 3-ethyl-2, 4-dimethylpyrrole, in the presence of TFA, first, then with 2,3-dichloro-5,6-dicyanop-benzoquinone (DDQ), and finally with boron tribromide to assemble the BODIPY 15. In parallel, we condensed the 3Hindole 6 with 2-chloromethyl-4-nitrophenol to produce the [1,3]oxazine 16a. After cleavage of the methoxy group with boron tribromide, we reacted the resulting compound 17a with 4-pentynoic acid, under the assistance of DCC and DMAP, to afford the [1,3]oxazine 18a. Finally, we coupled 15 and 18a, in the presence of sodium ascorbate (SA) and copper(II) sulfate, and condensed the resulting compound 19a with 4-dimethylamino benzaldehyde to generate the target compound 5a. Steady-State Absorption Spectroscopy. The steady-state absorption spectrum of the model photochrome 2a shows a band centered at 305 nm (a in Figure 6).22 Those of the model fluorophores 20 and 21 (Figure 7) instead reveal a bands at ca. 520 nm (c and d in Figure 6).21a Both absorptions can also be observed in the spectra of the fluorophore-photochrome dyads 3a-5a (a, c, and e in Figure 8), suggesting that their fluorescent and photochromic components have negligible ground-state interactions, even if they are integrated within the same molecular skeleton. Upon addition of acid, the [1,3]oxazine ring of the model photochrome 2a opens to generate 2c (Figure 9) with the concomitant appearance of a band at 569 nm (b in Figure 6) in the corresponding spectrum. Indeed, this transformation brings the 2-(4-dimethylaminophenyl)ethynyl appendage in conjugation with the 3H-indole heterocycle, and the resulting extended chromophore strongly absorbs in the visible region. A similar transformation occurs after addition of acid to the dyads 3a-5a. Once again, the [1,3]oxazine ring of their photochromic component opens under these conditions to generate 3c-5c (Figure 9). Consistently, the corresponding spectra reveal the appearance of the characteristic absorption of their extended 3H-indolium chromophores at ca. 560 nm (b, d, and f in Figure 8). In agreement with this interpretation, the spectra of the model fluorophore-photochrome dyads 19a (Figure 5) and 22a (Figure 8) remain essentially unchanged in the visible region after addition of acid (a-d in Figure S3, Supporting Information). In fact, both compounds lack the 2-(4-dimethylaminophenyl)ethynyl appendage on their photochromic component, and hence, the opening of their [1,3]oxazine ring cannot generate an extended 3H-indolium chromophore. Steady-State Emission Spectroscopy. The model fluorophores 20 and 21 emit at ca. 540 nm (e and f in Figure 6) upon excitation with quantum yields (φF in Table 1) of 0.40 and 0.62, respectively. After their incorporation within the fluorophorephotochrome dyads 3a-5a, these BODIPY fluorophores emit essentially in the same range of wavelengths (g, i, and k in Figure 8) but their φF are only 0.12, 0.07, and 0.02, respectively. Thus, the covalent attachment of the fluorescent components to the photochromic components results in partial quenching of their emission. Indeed, the redox potentials of 20, 21, and 4-dimethylaminostyrene (29 in Table S1, Supporting Information) suggest that the excited BODIPY components of the dyad 3a-5a can accept an electron from the 2-(4-dimethylaminophenyl)ethynyl appendage of the photochromic components. In fact,

Dyads Incorporating BODIPY and [1,3]Oxazine Components

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Figure 2. Photoinduced and reversible transformation of 2a-5a into 2b-5b.

this photoinduced electron transfer process is exoergonic in all instances with an estimated free energy change of ca. -0.5 eV.24 In agreement with this interpretation, the model dyads 19a and 22a, lacking the 2-(4-dimethylaminophenyl)ethynyl appendage, have significantly larger φF than 3a-5a (Table 1). The emission bands (e and f in Figure 6) of the models fluorophores 20 and 21 are positioned in the same range of wavelengths where the extended 3H-indolium chromophore of 2c absorbs (b in Figure 6). As a result, the excitation energy of these fluorophores can be transferred to the 3H-indolium chromophore, with a concomitant fluorescence quenching, if the two components are sufficiently close to each other. In addition, the redox potentials of these model compounds suggest that the excited BODIPY fluorophores can transfer an electron to the 3H-indolium cation of 2c, if sufficiently close to this component. In fact, this photoinduced electron transfer process is exoergonic in both instances with an estimated free energy change of -0.5 eV.24 Consistently, the emission intensity of 3a-5a (g, i, and k in Figure 8) decreases significantly after their transformation into 3c-5c (h, j, and l in Figure 8) with contrast ratios (χ in Table 1) of 4.5, 4.9, and 1.5, respectively. The ability of the 3H-indolium cation of 3c-5c to quench the emission of their BODIPY component is further confirmed by the behavior of the model compounds 19a, 22a, and 23-25 (Figures 5 and 7). The photochromic component of 19a and 22a lacks the 2-(4-dimethylaminophenyl)ethynyl group of 3a-5a. As a result, the opening of the [1,3]oxazine ring of 19a and 22a upon addition of acid is not accompanied by the appearance of the characteristic absorption of the extended 3Hindolium cation in the corresponding spectra (a-d in Figure S3, Supporting Information), and in fact, the emission intensity of their BODIPY fluorophore is essentially unaffected by this transformation (e-h in Figure S3, Supporting Information). Instead, the 3H-indolium chromophore is already in place within

22-24 and, consistently, the emission of the fluorescent component is almost completely suppressed in these systems (a-c in Figure S4, Supporting Information). Time-Resolved Absorption Spectroscopy. The absorption band at 305 nm (a in Figure 6) of the model photochrome 2a is associated with its 4-nitrophenoxy chromophore.22 In acetonitrile at 20 °C, laser excitation in the tail on this band results in the opening of the [1,3]oxazine ring with formation of 2b in less than 6 ns with a quantum yield of 0.07 (φP in Table 1). The photogenerated and zwitterionic isomer incorporates a 4-nitrophenoxy anion and a 3H-indolium cation, which absorb at 430 and 550 nm, respectively. As a result, the absorption spectrum of a solution of 2a, recorded 0.1 µs after excitation at 355 nm, reveals the characteristic bands of both chromophores (a in Figure 10). The model fluorophores 20 and 21 can also absorb at 355 nm (c and d in Figure 6). In fact, their excitation at this wavelength results in the bleaching of their S0 f S1 transitions (b and c in Figure 10), together with the appearance of bands at 430 and 400 nm, respectively, in the absorption spectra recorded 0.1 µs after laser irradiation. According to literature data,25 these bands can be assigned to absorptions of the BODIPY chromophores in the triplet manifold. The illumination of the fluorophore-photochrome dyads 3a and 5a at 355 nm results in the absorption of the exciting radiation by both chromophoric fragments. Consistently, the absorption spectra, recorded 0.1 µs after excitation, show the bleaching of the S0 f S1 transitions of the BODIPY fluorophore and bands for the photochromic component in the ring-opened state (d and f in Figure 10). Specifically, the band at 430 nm corresponds to a ground-state absorption of the 4-nitrophenolate chromophore of 3b and 5b and that at 560 nm is a groundstate absorption of the 3H-indolium fragment of these species. Thus, these observations demonstrate that the photochromic

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Figure 4. Synthesis of the fluorophore-photochrome dyad 4a.


component retains its photochemical behavior after coupling to the fluorescent component. However, the covalent attachment of the two components has a depressive effect on the quantum efficiency of the photoinduced ring-opening process. In particular, the [1,3]oxazine ring of 3a and 5a opens with φP of 0.05 and 0.02, respectively, while 2a has a φP 0.07. By contrast, the excitation of the fluorophore-photochrome dyad 4a does not produce the ring-opened isomer 4b under otherwise identical experimental conditions. In fact, its transient spectrum (e in Figure 10) resembles that of the model fluorophore 20 (b in

Figure 10) and does not reveal the ground-state absorptions of the ring-opened state of the photochromic fragments (a in Figure 10). The photogenerated isomer 2b has a lifetime of 2 µs (τ in Table 1) and reverts to the original species 2a with first-order kinetics.22 As a result, the transient absorptions at 430 and 550 nm decay monoexponentially with the thermal reisomerization of 2b back to 2a. The covalent connection of the photochromic component to the fluorescence component in the dyads 3a and 5a has negligible influence on their reisomerization kinetics. In all instances, the transient absorptions at 430 and 560 nm, corresponding to the 4-nitrophenolate anion and 3H-indolium cation of the photogenerated isomers 3b and 5b, decay monoexponentially on a microsecond time scale (a and b in Figure S5, Supporting Information). Specifically, curve fittings (c and d in Figure S5, Supporting Information) of the temporal absorbance profiles indicate τ of 3b and 5b to be 1 and 3 µs, respectively (Table 1). Thus, a full switching cycle can be completed within a few microseconds after excitation with both fluorophore-photochrome dyads In addition, both systems tolerate hundreds of cycles with no sign of degradation, even in the presence of molecular oxygen. Consistently, their steadystate and time-resolved absorption spectra do not change even after 400 excitation cycles. Time-Resolved Emission Spectroscopy. The steady-state absorption spectra (a and e in Figure 8) of 3a and 5a show that the band of their fluorescent component extends up to 600 nm. As a result, excitation at 532 nm (λON) is accompanied by the




characteristic BODIPY fluorescence. Similarly, the absorbance evolution (Figure S5, Supporting Information) at 560 nm upon excitation at 355 nm (λOFF) of 3a and 5a indicates that the [1,3]oxazine ring opens within the laser pulse to generate the corresponding isomers 3b and 5b. Thus, the simultaneous illumination of the sample at λON and λOFF with a single pulsed laser can be exploited to probe the emission of the BODIPY component integrated within the photogenerated isomers 3b and 5b. Relying on this irradiation protocol and a long-pass filter (>490 nm) to block and release λOFF, the influence of the

photochromic transformation on the emission intensity of the fluorescent component can be assessed. Indeed, the corresponding plots (a and b in Figure 11) demonstrate that the emission intensity at 580 nm for both fluorophore-photochrome dyads decreases significantly, as the long-pass filter is removed from the optical path to release the beam at λOFF, and returns to the original value, once the filter is mounted again in the original position. In fact, the fluorescence of both systems can be modulated for multiple switching cycles simply by blocking and releasing λOFF, consistently with the electron and energy transfer

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Figure 6. Steady-state absorption spectra (0.05 mM, MeCN, 20 °C) of 2a before (a) and after (b) addition of TFA (2 equiv), 20 (c), and 21 (d). Steady-state emission spectra (0.05 mM, MeCN, 20 °C) of 20 (e, λEx ) 437 nm) and 21 (f, λEx ) 447 nm).

TABLE 1: Photochemical and Photophysical Parametersa of the Fluorophore-Photochrome Dyads 3a-5a and Their Model Compounds 19-25 2a 3a 4a 5a 19a 20 21 22a 23 24 25

λEmb (nm)

φFb

χb,c

541 542 534 540 543 535 542 542 543 538

0.12 0.07 0.02 0.30 0.40 0.62 0.34 0.02 0.01 0.02

4.5 4.9 1.5 0.9

φP d

τe (µs)

0.07 0.05

2 1

0.02

3

0.9

a All parameters were measured in MeCN at 20 °C. b The emission wavelength (λEm), fluorescence quantum yield (φF), and contrast ratio (χ) were determined from the spectra in Figures 6, 8, S3 (Supporting Information), and S4 (Supporting Information). c χ is the ratio between φF measured before and that determined after addition of acid. d The quantum yield (φP) for the photochromic transformation was determined as described in the Supporting Information. e The lifetime (τ) of each photogenerated isomer was determined from the temporal absorbance evolutions in Figure S5 (Supporting Information).

processes designed into the photogenerated isomers 3b and 5b. In agreement with these operating principles, the emission intensity (c and d in Figure 11) of the model fluorophores 20 and 21, lacking the photochromic component, remains essentially unaffected under identical illumination conditions.

Figure 7. Model compounds 20-25.

Conclusions Photochromic oxazines can be connected to BODIPY fluorophores in 3-8 synthetic steps to generate fluorophorephotochrome dyads. In two of the three compounds synthesized, the photochromic fragment retains its photochemical properties, despite the presence of a BODIPY appendage. Specifically, the [1,3]oxazine ring opens upon ultraviolet excitation to generate a zwitterionic isomer in less than 6 ns with quantum yields of 0.02 and 0.05. In both instances, the photogenerated species reverts spontaneously back to the original one in a few microseconds. In addition, these photoswitchable systems are remarkably stable and tolerate hundreds of switching cycles with no sign of degradation. The selective excitation of the BODIPY component at visible wavelengths is accompanied by the emission of light in the form of fluorescence. However, the state of the photochromic component within the fluorophorephotochrome dyad regulates the excitation dynamics of the fluorescent fragment. In particular, the photogenerated state of


Figure 8. Steady-state absorption spectra (0.05 mM, MeCN, 20 °C) of 3a before (a) and after (b) addition of TFA (1 equiv), 4a before (c) and after (d) addition of TFA (1 equiv), and 5a before (e) and after (f) addition of TFA (5 equiv). Steady-state emission spectra (0.05 mM, MeCN, 20 °C) of 3a before (g, λEx ) 437 nm) and after (h, λEx ) 437 nm) addition of TFA (1 equiv), 4a before (i, λEx ) 425 nm) and after (j, λEx ) 425 nm) addition of TFA (1 equiv), and 5a before (k, λEx ) 447 nm) and after (l, λEx ) 447 nm) addition of TFA (5 equiv).


Figure 10. Absorption spectra (0.01-0.03 mM, MeCN, 20 °C) of 2a (a), 20 (b), 21 (c), 3a (d), 4a (e), and 5a (f) recorded 0.1 µs after laser excitation (355 nm, 6 ns, 15 mJ).

two photoswitchable dyads can be modulated with microsecond speeds by photoinducing the interconversion of the photochromic appendage. Hence, this protocol for fluorescence photoswitching with fast speeds and excellent fatigue resistance can evolve into the realization of valuable probes to overcome diffraction in fluorescence imaging and visualize biological samples with nanoscaled resolution. Experimental Procedures

Figure 9. Transformation of the [1,3]oxazines 2a-5a into the 3Hindolium cations 2c-5c upon addition of acid.

the photochrome incorporates a 3H-indolium cation, which can accept an electron or energy from the excited BODIPY and quench its fluorescence. Indeed, the emission intensity of the

Materials and Methods. Chemicals were purchased from commercial sources and used as received with the exception of MeCN, which was distilled over CaH2. Compounds 9, 20, 26a, and 30 were prepared according to literature procedures.21a,26 All reactions were monitored by thin-layer chromatography, using aluminum sheets coated with silica (60, F254). Fast atom bombardment mass spectra (FABMS) were recorded with a VG

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Deniz et al. and 532 nm (30 mJ) and the emission intensity was measured at 580 nm with and without a long-pass filter (>490 nm) in between laser and sample. Acknowledgment. We thank the the NSF (CAREER Award CHE-0237578 and CHE-0749840) and MIUR (PRIN 2008) for financial support. Supporting Information Available: Experimental procedures for the syntheses of 3a, 4a, 5a, 22a, and 25 and their precursors; steady-state absorption and emission spectra of 19a and 22a; redox potentials of 20, 21, 29, and 30; steady-state emission spectra of 23-25; absorbance evolution upon excitation of 3a and 5a; determination of the quantum yield for the photochromic transformations. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes

Figure 11. Emission intensity at 580 nm of solutions (0.01-0.03 mM, MeCN, 20 °C) of 3a (a), 5a (b), 20 (c), and 21 (d) recorded by turning on and off an exciting beam at λOFF (355 nm, 10 mJ, 6 ns), while illuminating the sample at λON (532 nm, 30 mJ, 6 ns).

Mass Lab Trio-2 spectrometer in a 3-nitrobenzyl alcohol matrix. High-resolution electrospray ionization mass spectra (HRESIMS) were recorded with an Agilent LCTOF spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded with Bruker Avance 400 and 500 spectrometers. Steady-state absorption spectra were recorded with a Varian Cary 100 Bio spectrometer, using quartz cells with a path length of 0.5 cm. Steady-state emission spectra were recorded with a Varian Cary Eclipse spectrometer in aerated solutions. Fluorescence quantum yields were determined with a fluorescein standard, following a literature protocol.27 Cyclic voltammograms were recorded with a CH Instruments 660 workstation in degassed MeCN solutions of Bu4NPF6 (0.1 M) under an atmosphere of Ar, using a glassy-carbon working electrode, a platinum counter electrode, and a Ag/AgCl reference electrode. Time-resolved absorption spectra and emission traces were recorded with a Luzchem Research mLFP-111 spectrometer in aerated solutions by illuminating orthogonally the sample with a Continuum Surelite II-10 Nd:YAG pulsed laser [pulse width ) 6 ns (fwhm)]. The laser pulse was probed with a fiber that synchronized the mLFP system with a Tektronix TDS 3032 digitizer operating in pretrigger mode. The signals from a compact Hamamatsu photomultiplier were initially captured by the digitizer and then transferred to a personal computer that controlled the experiment with Luzchem software developed in the LabView 5.1 environment from National Instruments. For absorption measurements, the laser was operated at 355 nm (10 mJ) and the transmittance was measured in the 350-700 nm spectral range. The monitoring beam was supplied by a ceramic xenon lamp and delivered through quartz fiber optical cables. For fluorescence measurements, the laser was operated simultaneously at 355 (10 mJ)

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(24) The free energy changes for the photoinduced electron transfer processes were estimated with the Rehm-Weller equation using the redox potentials reported in the Supporting Information (Table S1), the energy change for the S0 f S1 transitions of 20 and 21 (2.34 and 2.36 eV, respectively), and a fluorophore-photochrome separation of 0.5 nm. Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259–271. (25) Galletta, M.; Campagna, S.; Quesada, M.; Ulrich, G.; Ziessel, R. Chem. Commun. 2005, 4222–4224.

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JP107116D

Photoswitchable Fluorescent Dyads Incorporating BODIPY and [1,3

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