Boron Dipyrromethene As a Fluorescent Caging Group for Single

Letters pubs.acs.org/acschemicalbiology

Boron Dipyrromethene As a Fluorescent Caging Group for SinglePhoton Uncaging with Long-Wavelength Visible Light Nobuhiro Umeda,†,⊥ Hironori Takahashi,‡,⊥ Mako Kamiya,‡ Tasuku Ueno,† Toru Komatsu,†,§ Takuya Terai,† Kenjiro Hanaoka,† Tetsuo Nagano,† and Yasuteru Urano*,‡,∥ †

Graduate School of Pharmaceutical Sciences and ‡Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ∥ Basic Research Program, Japan Science and Technology Agency, 3-5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan S Supporting Information *

ABSTRACT: Caged compounds are useful tools for precise spatiotemporal modulation of cell functions, but in most cases uncaging requires ultraviolet (UV) light, which is cytotoxic and has limited tissue penetration. Therefore, caged compounds that can be activated by longer-wavelength light are required. Here we describe a novel photoelimination reaction of 4aryloxy boron dipyrromethene (BODIPY) derivatives and show that BODIPY can function as a caging group for phenol groups. We developed a novel BODIPY-caged histamine compound, which is photoactivatable with blue-green visible light to stimulate cultured HeLa cells in a spatiotemporally well-controlled manner. This caging strategy is expected to be widely applicable to develop tools for probing various cellular functions.

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with a blue-green visible light source (∼500 nm), we developed photoactivatable caged histamine, which can be used to stimulate living cells in a precisely controlled manner. We first synthesized 1,3,5,7-tetramethyl-BODIPY derivatives bearing aryl groups at the boron position according to the literature15 (1−7, Figure 1a and Supporting Information). We found that the fluorescence quantum efficiency (ϕfl) of these 4aryloxy BODIPY derivatives decreased as the HOMO energy level of the aryloxy groups increased, while the absorption/ emission wavelengths and the molar absorption coefficient (ε) varied only slightly (Figure 1c, Supplementary Table S1). Since it has been reported that BODIPY fluorescence can be quenched by photoinduced electron transfer (PeT) from adjacent aryl groups,16 the quenching of these 4-aryloxy BODIPY derivatives is also considered to be due to PeT from the 4-aryloxy groups. During these spectroscopic investigations, we serendipitously discovered the very intriguing phenomenon that some of these 4-aryloxy BODIPY derivatives were photocleavable at the 4-position by visible light irradiation. Indeed, we confirmed by HPLC analysis and NMR characterization that solvolyzed BODIPY and a hydroxy aryl compound were produced after irradiation with visible light

aged compounds have been widely used as a biological tool for investigating or perturbing cellular dynamics/ functions of living cells in a spatiotemporally controlled manner. However, uncaging of conventional caged compounds by single photon excitation generally requires ultraviolet (UV) to violet light (250−400 nm),1,2 which is cytotoxic to living cells3 and has low tissue penetration. Therefore, caged compounds that can be activated by longer-wavelength light have been desired. Despite massive efforts to develop such caged compounds, most suffer from drawbacks, such as a requirement of additional reagents4 or lack of evidence for practicality as caged compounds in living systems.5,6 Amineruthenium complexes can be photolyzed by visible wavelengths up to 532 nm to induce cellular response,7−9 but their relatively broad absorption spectrum hampers fluorescence observation using fluorescent indicators such as Rhod-2, and there remain concerns about potential photodamage, since they are originally based on photosensitizers.10 Recently, blue-cyan light-absorbing caging groups based on coumarin derivatives have also been reported.11,12 Here, we focused on the widely known boron dipyrromethene (BODIPY) fluorophore,13,14 since it has a sharp and intense absorption profile that might minimally occupy the wavelengths needed for fluorescence observation and shows negligible triplet-state formation, which should guarantee low phototoxicity and high biocompatibility. On the basis of our serendipitous finding that some 4-aryloxy BODIPY derivatives undergo photoelimination reaction under irradiation © XXXX American Chemical Society

Received: July 1, 2014 Accepted: August 20, 2014

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dx.doi.org/10.1021/cb500525p | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

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Figure 1. Photoelimination reaction of 4-aryloxy BODIPY derivatives. (a) General scheme of the photoreaction. (b) Time course of BDP-7HC (4) consumption and photoproducts production upon irradiation. Irradiation: 475−490 nm, 15 mW cm−2. (c and d) Fluorescence quantum efficiency (ϕfl) and uncaging quantum efficiency (ϕu) of 4-aryloxy BODIPY derivatives are plotted against HOMO energy level of the corresponding phenols.

Figure 2. Development of BODIPY caged histamine. (a) Scheme of uncaging reaction of BcHA-1 (13) and -2 (18). (b) Cellular distribution of 1 μM BcHA-1 and 5 μM BcHA-2 in HeLa cells. Scale bars, 100 μm. (c) Absorption (black solid line) and fluorescence (red solid line) spectra of BcHA-2. (d) Time course of BcHA-2 consumption and histamine production. Irradiation: 460−500 nm, 29 mW cm−2. Data are presented as mean ± SEM from three independent experiments. (e) Dose−response curves of BcHA-2 and histamine. Data are presented as mean ± SEM, n > 40 cells for each condition from two independent experiments.

(475−490 nm) (Figure 1a,b and Supplementary Figures S1 and S2). More precisely, the efficiency of photocleavage (ϕu) of 4aryloxy BODIPY derivatives was greatly dependent on the HOMO energy level of the aryl group; the derivatives whose ϕfl was low due to the quenching via PeT were efficiently decomposed with high ϕu upon irradiation with blue-green light (Figure 1d, Supplementary Table S1). This inverse correlation between ϕfl and ϕu suggests that the uncaging reaction of the BODIPY caging group proceeds via a PeT process in which a charge-separation intermediate (CS state) involving the cation radical of the aryl group and anion radical of the fluorophore is produced, followed by solvolysis of the B−

O bond of the positively charged aryl group. As the previous report suggested that cationization can greatly reduce the pKa of the hydroxy aryl groups,17 we speculated that production of the CS state would be an important determinant of photocleavage of the BODIPY caging group. We also found that the PeT-based quenching of fluorescence is a necessary but not sufficient condition for efficient uncaging because a decrease of ϕu above −0.2 hartree was observed, which might result from a faster back electron transfer (BeT) process, shortening the lifetime of the CS state.18 Concerning the values of uncaging quantum efficiency (ϕu), the values of our BODIPY derivatives are lower than those of known UVB



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excitable caging groups (Supplementary Table S1).2 However, this can be compensated by the higher molar absorption coefficient (ε) of BODIPY derivatives, since the product of ε and ϕu is as high as 100. Indeed, as described later, we demonstrated that these values are sufficient for practical usage to uncage a physiological concentration of bioactive molecules with a conventional laser setup for fluorescence microscopy. Further, uncaging with even longer-wavelength light was accomplished based on another π-extended BODIPY derivative, dihydronaphtho BODIPY (DHNB), which shows absorption at over 600 nm (8 and 9, Supplementary Figure S3 and Supplementary Table S2),19 by introducing an appropriate aryloxy group on the boron atom to quench the fluorescence of DHNB. To our knowledge, this is the first report of a caging group activated by single-photon excitation at a wavelength over 600 nm. Next, in order to investigate biocompatibility, we aimed at developing biofunctional caged compounds based on our BODIPY derivatives. Due to the lower ϕu of DHNB derivatives compared to that of 1,3,5,7-tetramethyl-BODIPY derivatives, we decided to design caged compounds based on the latter. As a target molecule, we chose histamine, which is a biogenic amine that plays an important role in various physiological and pathophysiological processes, including sleep-wakefulness, allergy, and inflammation.20,21 In the molecular design of caged histamines (BcHAs), the terminal amine group of histamine was caged with BODIPY through a benzyloxycarbonyl linker, so that the phenolate group released upon light irradiation further decomposes to produce free histamine together with CO2 and quinone methide, which is immediately hydrolyzed to generate 4-hydroxybenzyl alcohol22 (Figure 2a). We first developed BcHA-1 (13) based on conventional 1,3,5,7-tetramethyl BODIPY (Table 1). Although it exhibited

derivatization using o-phthalaldehyde and thiol (Figure 2d).24 While BcHA-2 was consumed completely after sufficient light irradiation, histamine was released in a maximum yield of 40%, probably owing to a photoinduced side reaction, though this has not yet been clarified. We also confirmed that the agonist activity of BcHA-2 for histamine H1 receptor was greatly suppressed compared to that of histamine by measuring the fluorescence change of Rhod 2-AM, a Ca2+ indicator, which was preloaded into HeLa cells (Figure 2e). Further, BcHA-2 showed no antagonist activity toward H1 receptor at the maximum experimental concentration of 5 μM (Supplementary Figure S5). Next, we tested whether single-photon uncaging of BcHA-2 can induce an increase of intracellular Ca2+ in HeLa cells. Light irradiation via a confocal laser scanning microscope equipped with argon lasers successfully evoked a Ca2+ response (Figure 3a, top row). Cells showed no response in the control

Table 1. Spectroscopic and Photochemical Properties of BcHA-1 and BcHA-2 BcHA-1 (13) BcHA-2 (18)

λabsa (nm)

λema (nm)

ϕfla

ϕu

498 510

511 524

0.13 0.11

3.0 × 10−4a 3.9 × 10−4b

a

Measured in 0.1 M sodium phosphate buffer pH 7.4. bMeasured in 0.2 M sodium phosphate buffer pH 7.4.

good photocleavability in vitro, it failed to stimulate cultured HeLa cells (Supplementary Figure S4). There are two possible reasons for this failure: the low release of histamine or mismatched localization of the caged compound and target receptor. Since even prolonged photoirradiation did not induce a cellular response, we considered that the latter was more likely. Therefore, we examined the cellular localization of BcHA-1 by means of fluorescence microscopy, utilizing the suppressed BODIPY-derived fluorescence. As a result, we found that BcHA-1 tends to localize in membranous structures in cells, presumably due to its hydrophobic nature (Figure 2b, left). Considering that the histamine H1 receptor exists on the cell surface, we considered that we needed a more hydrophilic derivative, which should be localized in the extracellular space. Therefore, we newly designed BcHA-2 (18) (Table 1), based on more hydrophilic 2,6-dicarboxy-1,3,5,7-tetramethyl BODIPY (dicarboxy BODIPY),23 which was indeed localized in the extracellular medium (Figure 2b, right). We also confirmed that BcHA-2 is photodecomposed to produce histamine in a lightdependent manner by HPLC analysis after fluorescent

Figure 3. Application of BcHA-2 to HeLa cells. (a) Uncaging experiments of BcHA-2. BcHA-2 and pyrilamine were used at 5 and 1 μM, respectively. Scale bar, 100 μm. (b) Statistical analysis of data in panel a. Mean ± SEM, *** p < 0.001, Games-Howell method, n = 30 cells from two independent experiments. (c) Representative traces of intracellular Ca2+ dynamics in Supplementary Figure S7b.

conditions, and the Ca2+ response was inhibited in the presence of pyrilamine, an H1-antagonist (Figure 3a,b and Supplementary Figure S6),25 indicating that HeLa cells respond specifically to histamine produced by uncaging of BcHA-2. We also attempted uncaging of BcHA-2 to specifically stimulate cells in a small or broad region of interest. In the presence of BcHA-2, light irradiation evoked a cellular response in a small square region located in the center of the field of view of the confocal laser scanning microscope (Supplementary Figure S7a and Movie S1). The response subsequently extended outside the irradiated area, presumably due to the diffusion of released C



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JSPS Core-to-Core Program, A. Advanced Research Networks, as well as The Daiichi-Sankyo Foundation of Life Science and The Naito Foundation Natural Science Scholarship (grant to Y.U.), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (grant to M.K.), The Tokyo Society of Medical Sciences (grant to M.K.), and a JSPS stipend to N.U.

histamine. Then, we also tried to uncage BcHA-2 over a broader area (a quarter of the field of view). As a result, we succeeded in inducing a cellular response in a broad area, demonstrating that BcHA-2 would serve as a tool for modulating cellular functions even over broad areas (Supplementary Figure S7b and Movie S2). Further, the fluorescence traces of Rhod 2-AM exhibited characteristic intracellular Ca2+ increase and oscillation in several cells (Figure 3c), in accordance with the previous report that histamine stimulation results in periodic Ca2+ increases.26 We also confirmed that the stimulation level is adjustable by changing the laser output (Supplementary Figure S8). In conclusion, we describe novel BODIPY-based caging groups that undergo single-photon uncaging with longerwavelength visible light, which should be available as versatile caging groups for a wide variety of bioactive molecules. We believe that our BODIPY-caged compounds offer two major advantages for practical biological applications. The first is the ability to uncage in response to longer-wavelength light with a sharp absorption profile, which would not interfere with the wavelengths required for fluorescence observation. Furthermore, since we succeeded in single-photon uncaging of DHNB derivatives with orange-red light over 600 nm, it should be feasible to develop caged compounds activatable with much longer-wavelength light, which is impossible with established caging groups. The second key advantage of BODIPY-caged compounds is that the localization of the caged compounds can be visualized by utilizing the intrinsic fluorescence of BODIPY. This is practically important for biological applications, because the distribution of a caged compound greatly affects its efficacy as shown above. From these points of view, our BODIPY-caged compounds offer a chemical basis for designing caged compounds activatable with visible light and should be useful for precise perturbation of cellular functions, not only in cultured cells but also in living tissues or animals. Work along this line is in progress.

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METHODS

The details of the methods are provided in the Supporting Information.

ASSOCIATED CONTENT

* Supporting Information S

Supplementary figures, tables, and movies and details of the methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research (KAKENHI), grants 20117003 and 23249004 to Y.U., grants 23113504, 25113707, 25870180 to M.K., and grant 25104506 to T.T.), a Specially Promoted Research Grant (22000006 to T.N.), by D



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(17) Bordwell, F. G., and Cheng, J. (1991) Substituent effects on the stabilities of phenoxyl radicals and the acidities of phenoxyl radical cations. J. Am. Chem. Soc. 113, 1736−1743. (18) Miura, T., Urano, Y., Tanaka, K., Nagano, T., Ohkubo, K., and Fukuzumi, S. (2003) Rational design principle for modulating fluorescence properties of fluorescein-based probes by photoinduced electron transfer. J. Am. Chem. Soc. 125, 8666−8671. (19) Chen, J., Burghart, A., Derecskei-Kovacs, A., and Burgess, K. (2000) 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes modified for extended conjugation and restricted bond rotations. J. Org. Chem. 65, 2900−2906. (20) Thakkar, M. M. (2011) Histamine in the regulation of wakefulness. Sleep Med. Rev. 15, 65−74. (21) O’Mahony, L., Akdis, M., and Akdis, C. A. (2011) Regulation of the immune response and inflammation by histamine and histamine receptors. J. Allergy Clin. Immunol. 128, 1153−1162. (22) Dyer, R. G., and Turnbull, K. D. (1999) Hydrolytic stabilization of protected p-hydroxybenzyl halides designed as latent quinone methide precursors. J. Org. Chem. 64, 7988−7995. (23) Komatsu, T., Urano, Y., Fujikawa, Y., Kobayashi, T., Kojima, H., Terai, T., Hanaoka, K., and Nagano, T. (2009) Development of 2,6carboxy-substituted boron dipyrromethene (BODIPY) as a novel scaffold of ratiometric fluorescent probes for live cell imaging. Chem. Commun. 45, 7015−7017. (24) Lindroth, P., and Mopper, K. (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal. Chem. 51, 1667−1674. (25) Hazama, A., Yada, T., and Okada, Y. (1985) HeLa cells have histamine H1-receptors which mediate activation of the K + conductance. Biochim. Biophys. Acta 845, 249−253. (26) Sauvé, R., Diarra, A., Chahine, M., Simoneau, C., Morier, N., and Roy, G. (1991) Ca2+ oscillations induced by histamine H1 receptor stimulation in HeLa cells: Fura-2 and patch clamp analysis. Cell Calcium 12, 165−176.

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Boron Dipyrromethene As a Fluorescent Caging Group for Single

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