Generation of Triplet Excited States via Photoinduced Electron Transfer in meso- anthra-BODIPY: Fluorogenic Response Towards Singlet Oxygen in Solution and in Vitro

Heavy  atom‐free  BODIPY‐anthracene  dyads (BADs) upon photoexcitation generate  locally excited triplet excited states by way of photoinduced electron transfer (PeT), followed by recombination of the resulting charge‐separated states  (CSS).  Subsequent  quenching  of  the  triplet  states  by molecular oxygen produces singlet oxygen (1O2), which reacts with  the anthracene moiety yielding highly  fluorescent  spe‐ cies.  The  steric  demand  of  the  alkyl  substituents  in  the BODIPY  subunit defines  the  site of O2 addition. Novel bis‐ and tetraepoxides along with bicyclic acetal products arising from a chain of rearrangements of anthracene endoperoxides were  isolated and characterized. O2 generation by BADs  in living cells provides fluorescent visualization of the dyads dis‐ tribution promising new imaging applications. Optical  probes  based  on  photoinduced  electron  transfer (PeT) in donor‐acceptor dyads have found broad use in diag‐ nostics, particularly for the detection of biomolecules, metal ions, reactive oxygen species (ROS) and measurement of  in‐ tracellular pH.1 The PeT process  leads  to  formation of non‐ emissive charge‐separated  states  (CSS) which decay back  to the ground state via different pathways. Among  those  is re‐ combination of CSS, which may lead to locally excited triplet states in the molecule.2 Recently this process has attracted at‐ tention as a method to increase intersystem crossing without directly relying to the heavy atom effect.3 The possibility for singlet oxygen (O2) generation by donor‐ acceptor dyads mediated by PeT has not been realized so far in practical sense. It could be expected that O2 generation by PeT‐based optical probes    in biological environments would affect their optical response and simultaneously induce cyto‐ toxicity. This  is of special concern  in the case of ROS detec‐ tion, where sensitization of O2 by the probe itself may lead to false  positives  and  incorrect  interpretations.4 On  the  other hand, PeT‐mediated O2 generation could provide a new tool for theranostic applications, since the process of charge sepa‐ ration can be turned on/off by various stimuli. Herein we re‐ port  readily accessible heavy atom‐free BODIPY‐anthracene dyads  (BADs)  that  can  act  as  efficient  triplet  sensitizers, providing fluorescent response towards generated O2. While a number of triplet sensitizers based on halogenated BODIPYs have been reported during the last decade,5 obser‐ vations of  triplet excited state  formation  in heavy atom‐free BODIPYs are rare.6 In our search into efficient donor‐acceptor PS, we have focused on BADs 1 and 2 (Scheme 1). BODIPYs are known  to  be  efficient  energy  and  electron  acceptors when combined with anthracene.7 Although compound BAD1 has been  reported  to  exhibit  PeT,  no  triplet  excited  states  for‐ mation has been noted.8

(BADs) upon photoexcitation generate locally excited triplet excited states by way of photoinduced electron transfer (PeT), followed by recombination of the resulting charge-separated states (CSS). Subsequent quenching of the triplet states by molecular oxygen produces singlet oxygen ( 1 O2), which reacts with the anthracene moiety yielding highly fluorescent species. The steric demand of the alkyl substituents in the BODIPY subunit defines the site of 1 O 2 addition. Novel bisand tetraepoxides along with bicyclic acetal products arising from a chain of rearrangements of anthracene endoperoxides were isolated and characterized. 1  Optical probes based on photoinduced electron transfer (PeT) in donor-acceptor dyads have found broad use in diagnostics, particularly for the detection of biomolecules, metal ions, reactive oxygen species (ROS) and measurement of intracellular pH. 1 The PeT process leads to formation of nonemissive charge-separated states (CSS) which decay back to the ground state via different pathways. Among those is recombination of CSS, which may lead to locally excited triplet states in the molecule. 2 Recently this process has attracted attention as a method to increase intersystem crossing without directly relying to the heavy atom effect. 3 The possibility for singlet oxygen ( 1 O 2 ) generation by donoracceptor dyads mediated by PeT has not been realized so far in practical sense. It could be expected that 1 O 2 generation by PeT-based optical probes in biological environments would affect their optical response and simultaneously induce cytotoxicity. This is of special concern in the case of ROS detection, where sensitization of 1 O 2 by the probe itself may lead to false positives and incorrect interpretations. 4 On the other hand, PeT-mediated 1 O 2 generation could provide a new tool for theranostic applications, since the process of charge separation can be turned on/off by various stimuli. Herein we report readily accessible heavy atom-free BODIPY-anthracene dyads (BADs) that can act as efficient triplet sensitizers, providing fluorescent response towards generated 1 While a number of triplet sensitizers based on halogenated BODIPYs have been reported during the last decade, 5 observations of triplet excited state formation in heavy atom-free BODIPYs are rare. 6 In our search into efficient donor-acceptor PS, we have focused on BADs 1 and 2 (Scheme 1). BODIPYs are known to be efficient energy and electron acceptors when combined with anthracene. 7 Although compound BAD1 has been reported to exhibit PeT, no triplet excited states formation has been noted. 8

Scheme 1. Photoinduced transformations of BADs.
Upon broad-band visible light irradiation of air-saturated solutions of BAD1 in a range of polar solvents we observed, to our surprise, completely selective formation of BAD1-BE, which could be isolated in 5% yield along with recovered unreacted starting material (Scheme 1). In contrast, irradiation of BAD2 under the same conditions resulted in complete conversion of the substrate with formation of two products, bicyclic acetal derivative (BAD2-BA) and tetraepoxide (BAD2-TE), which were isolated in 80% and 10% yields, respectively. The structures of the products were confirmed by NMR spectroscopy and X-ray crystallography (for details see Supporting Information (SI)). Unlike BADs 1 and 2, isolated compounds exhibit bright fluorescence independent of the solvent polarity. For instance, the emission quantum yields of BAD1-BE in CH 2 Cl 2 and hexane were determined to be 0.91 and 0.89, respectively.
The formation of these products appears to be due to the sensitization of oxygen and subsequent [4+2] cycloaddition of the resulting 1 O 2 , which is typical for anthracene derivatives. 9 Singlet oxygen quantum yields of BADs were measured using 1,3-diphenylisobenzofuran as 1 O2 trap, giving values of 0.67 and 0.38 in ethanol, for BAD1 and BAD2, respectively. In order to understand the mechanism of 1 O 2 formation we studied the excited state dynamics of the dyad BAD1 by broadband Vis-NIR sub-pico-to microsecond transient absorption (TA) pump-probe spectroscopy. . c) ns-µs Transient absorption spectra of degassed BAD1 solutions following excitation at 355 nm by 700 ps laser pulses. The spectra were integrated from 3-5 ns (black line), 10-100 ns (red line), 0.1-1 µs (green line), 1-5 µs (blue line), and 10-100 µs (cyan line). d) Kinetics observed for the bands at 570 nm and 680 nm, assigned to the BODIPY triplet state and anthracene radical-cation, respectively, in the absence and presence of oxygen (solid and dotted lines, respectively).
Immediately after photoexcitation with fs pulses at 355 nm a broad band around 360 nm due to the anthracene's singlet excited state (S 1 ) absorption, which partially overlaps with the ground state photo bleach (PB), was observed ( Figure 1a). The concomitant decay of this band and simultaneous rise of the bleach at 505 nm indicate ultrafast energy transfer (EnT) from anthracene to BODIPY subunit, populating the singlet state of the latter, S BDP . Furthermore, another absorption band grows at 580 nm, and it was assigned to the BODIPY radical-anion (BDP -• ), 10 forming due to the PeT process. This band rises during the first 100 ps, simultaneously shifting to 570 nm, indicating a transition of the radical-anion to another excited state (see inset of Figure 1a). Synchronously with the rise of the BDP -• absorption (580 nm), yet another absorption band, centered at 680 nm rises, presumably due to the anthracene rad-ical-cation (Ant +• ), in line with previous reports. 1 1 Global fitting of the PB decays at 380 nm and 400 nm and the rise of BDP -• and Ant +• bands (Figure 1b) yields time constants of 1.15 ps and 0.54 ps for the EnT and PeT processes, respectively.
In the ns-μs TA experiments, a rise of an absorption band at 570 nm over 1 µs was observed, indicating formation of longlived states (Figure 1c). Previous reports on the TA spectra of BODIPY support the assignment of this band to the BODIPY triplet state (T BDP ) absorption. 10b The band at 570 nm was quenched and decayed faster in the presence of oxygen (Figure 1d). In contrast, the anthracene radical cation-absorption band at 680 nm was impacted by oxygen significantly less. The T BDP lifetime in the absence of O 2 was determined to be 41 µs. The observed transition from the bands originating in CSS to the absorption by the triplet suggests that the formation of CSS is a prerequisite for populating of T BDP . The frontier molecular orbitals diagram (Figure 2a) shows that the two highest occupied orbitals π ant and π BDP located on the anthracene and BODIPY subunit, respectively, are nearly degenerate. Density functional theory (DFT) calculations on these molecules (see SI for computational details) confirm that in BAD1 PeT could take place from π ant to singly occupied π BDP thus leading to singlet charge transfer state S CSS that is 0.4 eV more stable than the S BDP excited state. Unlike the valence excited states, CSS has a very low ferromagnetic exchange coupling integral due to negligible overlap of singly occupied orbitals π ant and π* BDP located in mutually orthogonal molecular moieties thus leading to a very small singlet-triplet energy gap (S-T gap). Two pathways for triplet state generation from CSS may yield the lowest local T BDP state ( Figure  2b): spin-orbit charge transfer intersystem crossing (SOCT-ISC) and radical pair intersystem crossing (RP-ISC) ), followed by triplet charge recombination. 12 As has been shown in extensive works of Wasielewski and co-workers, 13 SOCT-ISC prevails for systems with strong electronic couplings, requiring short distances between the subunits (4.3 Å in BAD1). On the other hand, due to the small S-T gap in the RP state, mixing of S CSS and T CSS states is possible due to e.g. electron-nuclear hyperfine coupling. More detailed studies will be necessary to distinguish between mechanisms governing spin interconversion in BADs.
The observed PeT process is clearly manifested in the spectroscopic properties of BADs. The fluorescence of the BODIPY is quenched in polar solvents as evidenced by the negligible values of Φ f observed, compared to the strong emission in non-polar solvents (Table S3). A broad emission band at 610 nm was observed in polar solvents. Such red-shifted broad emission bands arising from charge transfer excited states were reported for various donor-acceptor systems. 14 DFT calculations in vacuo show that S CSS state is approximately 0.2 eV higher in energy than the valence S BDP state. The dipole moment for the S CSS state was computed to be μ = 19 D in vacuo, which is much higher than that for the valence S BDP state (5 D). Interactions of CSS with polar solvent result in a decrease of the S CSS state dipole moment to 1.1 D and change the relative energy ordering of the S BDP and S CSS states, making PeT process favourable.  The formation of bicyclic acetal and tetraepoxide products from BAD2 is likely to take place via an 9,10-endoperoxide intermediate (Scheme 2). The rearrangement of endoperoxides into bisepoxides can be induced either thermally or photochemically. 9 The process is caused by the homolytic cleavage of the peroxide O-O bond, followed by rearrangement to more stable bisepoxides. Commonly such bisepoxides, containing a cyclohexadiene ring, could not be isolated, but only trapped with dienophiles. 15 Indeed, we found no traces of this intermediate in the reaction mixture. According to previous reports, the formation of a bicyclic acetal from bisepoxide may take place via heterolytic cleavage of the epoxide C-C bond, leading to an ylide-type bipolar intermediate. 15b This is then followed by C-O bond rupture of a second epoxide fragment, leading to rearomatization of the lateral ring and formation of the acetal bridge. The rearrangement competes with addition of 1 O 2 molecule to the diene moiety leading to BAD2-TE.
In contrast, the bisepoxide BAD1-BE is stable and showed no formation of the rearrangement products. Its formation likely proceeds via the mechanism discussed above, involving O-O homolytic cleavage and further isomerization. The addition of 1 O 2 to the outer ring in this case is surprising, as the central 9,10-site is the most reactive, based on frontier molecular orbital analysis. The influence of steric factors on the regioselectivity of endoperoxide formation has previously been reported for acenes with bulky substituents at the ortho-positions of the aryl groups. 16 Comparison with BAD2 shows that the unusual reactivity of BAD1 can be attributed to the effect of methyl substituents in position 4 of the BODIPY core. This can be seen in the XRD data where C-4 methyl substituents in BAD1 are forming a steric like shield of the C-9 position of the anthracene unit. Introduction of methyl groups into the BODIPY pyrrole rings shields the inner ring of the orthogonal anthracene residue, making the approach of 1 O 2 molecule difficult. Different reactivity of BADs towards 1 O 2 accounts for the variations in their fluorescence response (Figure 3b) due to the cycloaddition to the anthracene moiety, which takes place considerably faster for BAD2.
The rise of BAD2 fluorescence due to cycloaddition reaction is manifested even at 1 µM concentration, and it reaches the intensities comparable to those of the emission of a strongly fluorescent reference BODIPY compound (Fig. S7). It was of special interest for us to investigate whether the sensitization process can be reproduced in live cells. For this purpose we generated appropriate water-soluble derivatives. Substitution of fluorine atoms with N,N-dimethylaminopropyne-1 residues gave corresponding BADs 3 and 4. Quaternization of the dimethylamino group with 1,3-propanesultone then gave BADs 5 and 6, bearing zwitterionic fragments (betaine) which imparted the desired aqueous solubility.
To examine the fluorescence response of BADs 5 and 6 towards self-sensitized 1 O 2 in cells, human breast cancer (MDA-MB-468) cells were incubated with BADs 5 and 6 (1 µM) followed by irradiation with broadband visible light (400 -700 nm, 23.8 mWcm -2 ). Cells were irradiated for 0, 2.5 and 5 minutes and were visualised by confocal fluorescence microscopy. Over the time course of irradiation an increase in the fluorescence intensity was observed for BAD6 ( Figure 4) indi-cating firstly, that the chromophore had entered the cells, rather than simply associating with the external cell membrane; and secondly, that the fluorescence increased in a similar way to that observed for BAD2 in homogeneous solution. However, this behavior was not replicated in the case of BAD5, which showed no observable fluorescence on this timescale, even when irradiated with the higher light doses. Lower fluorogenicity of BAD5 is in accord with the behaviour of parent BAD1, which was shown to react with 1 O 2 considerably slower than BAD2. At higher concentrations of BADs evidence of morphological changes to the cells upon irradiation, most noticeably "blebbing" of the cell membrane, was observed (Fig. S12), indicating apoptotic behaviour. Cell viabilities after incubation with a range of concentrations (1 -50 M) of BADs 5 and 6, followed by light treatment (23.8 mWcm -2 ), were assayed by MTT protocol. The results obtained indicate that both watersoluble BADs induce a significant cytotoxic effect on the cells, whereas negligible cytotoxic effects were observed in the control group under otherwise identical conditions, but without irradiation (Fig. S13). Median lethal doses (LD 50 ) of BADs were found to be 4 M, thus the lower dose of 1 M was selected for imaging experiments.
In conclusion, we have demonstrated that heavy atom-freedonor-acceptor dyads can be used as 1 O 2 sensitizers, whereby the triplet excited states playing the key role in oxygen sensitization form by way of photoinduced electron transfer. Moreover, the described dyads are capable of forming strongly fluorescent species with self-sensitized 1 O 2 in biological media. The fluorescent response allows visualization of 1 O 2 formation within the cells and, consequently, fine-tuning of the photon doses required to cause oxidative stress. These sensitizers may give rise to a promising new class of materials for photonic applications which depend on triplet excited states generation. Studies to extend the scope such systems are underway.

Supporting Information
Synthetic procedures, NMR, optical spectra, computational details, X-ray crystallographic data for BAD1, BAD1-BE, BAD2-BA, BAD2-TE in CIF format, fluorescence microscopy images and cytotoxicity assay protocols. This material is available free of charge via the Internet at http://pubs.acs.org.