Angewandte Communications Chemie

International Edition:DOI:10.1002/anie.201705803 Bioimaging German Edition:DOI:10.1002/ange.201705803 AHighly Efficient ChemiluminescenceProbe for the Detection of Singlet Oxygen in Living Cells Nir Hananya+,Ori Green+,Rachel Blau, Ronit Satchi-Fainaro,* and Doron Shabat*

[9] 1 Abstract: Singlet oxygen is among the reactive oxygen species extremely weak. Explicitly,for the imaging of O2,the (ROS) with the shortest life-times in aqueous media because of advantage of chemiluminescence over is even its extremely high reactivity.Therefore,designing sensors for more substantial, since the used for excitation of the dye 1 1 [10] detection of O2 is perhaps one of the most challenging tasks in could induce O2 generation by itself. the field of molecular probes.Herein, we report ahighly Several years ago,McNeill and co-workers reported 1 [11] selective and sensitive chemiluminescence probe (SOCL-CPP) a“trap-and-trigger” chemiluminescent probe for O2. 1 1 [12] for the detection of O2 in living cells.The probe reacts with O2 They used the enol-ether precursor of Schaapsdioxetane 1 to form adioxetane that spontaneously decomposes under to trap the O2 at the first step.Inasecond step,the dioxetane physiological conditions through achemiexcitation pathway to chemiluminescence was triggered by the addition of fluoride. emit green light with extraordinary intensity. SOCL-CPP In this system, chemiluminescence measurements were lim- demonstrated promising ability to detect and image intra- ited to organic solution owing to the quenching of the 1 [13] cellular O2 produced by aphotosensitizer in HeLa cells during emitting species by water. We therefore speculated that (PDT) mode of action. Our findings adioxetane that efficiently emits light in aqueous solution make SOCL-CPP the most effective knownchemilumines- could be utilized to develop achemiluminescent probe that is 1 1 cence probe for the detection of O2.Weanticipate that our capable of real-time monitoring of O2 production under 1 chemiluminescence probe for O2 imaging would be useful in physiological conditions. 1 PDT-related applications and for monitoring O2 endoge- Recently,wereported astriking substituent effect for nously generated by cells in response to different stimuli. Schaapschemiluminescence dioxetanes,obtained through the incorporation of an electron withdrawing group at the Reactive oxygen species (ROS) have an increasingly recog- ortho-position of the phenol.[14] These new dioxetane lumino- nized role in cell signaling and stress response.[1] Among all phores were able to emit light under aqueous conditions with 1 [2] ROS, singlet oxygen ( O2)isgetting substantial attention, an intensity 3000-fold greater than that of the original especially in view of the recent progress in the field of Schaapsdioxetane.Wesought to use the enol-ether pre- photodynamic therapy (PDT) for cancer treatment.[3] There- cursor of our highly bright dioxetane luminophores as 1 1 fore,real-time monitoring of O2 under biologically relevant achemiluminescent probe for the detection of O2.Herein, conditions is of great interest. Although the we report anew highly efficient chemiluminescence probe for 1 [4] of O2 at awavelength of 1270 nm can be directly observed, detection of singlet oxygen under physiological conditions. 1 1 it is very challenging to detect small amounts of O2 by such Our strategy for real-time monitoring of O2 by chem- methods because of the extremely low quantum yield under iluminescence is presented in Figure 1. Probe SOCL (singlet [5] 1 aqueous conditions. Alternative approaches were aimed to oxygen chemiluminescence) reacts with O2 to generate 1 [6] develop small-molecule reaction-based probes for O2 ; aphenol-dioxetane species.This species spontaneously among them, fluorescent turn-on probes have appeared as decomposes in water (t1/2 = 10 min) through achemiexcitation the most sensitive.[7] Several fluorescent probes were also process to produce the corresponding electronically excited 1 [8] suitable for the imaging of O2 in living cells. In general, benzoate ester.The decay of the latter to its ground-state is chemiluminescence imaging might be advantageous over accompanied by the emission of highly bright green light. In fluorescence imaging,since in chemiluminescence there is no our design, the role of the acrylic acid substituent at the ortho need for light irradiation, and thus background signal is

[*] N. Hananya,[+] O. Green,[+] Prof. D. Shabat School of Chemistry,Faculty of Exact Sciences, TelAviv University TelAviv 69978 (Israel) E-mail:[email protected] R. Blau, Prof. R. Satchi-Fainaro Department of Physiologyand Pharmacology,Faculty of Medicine, TelAviv University TelAviv 69978 (Israel) E-mail:[email protected] [+]These authors contributed equally to this work. Supportinginformation and the ORCID identification number(s) for 1 the author(s) of this article can be found under: Figure 1. Structure of O2 chemiluminescent probe SOCL and its 1 https://doi.org/10.1002/anie.201705803. chemiexcitation pathway upon reaction with O2.

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position of the phenol is to form adonor–acceptor pair that comparison and LOD calculations). Thebetter sensitivity increases the emissive nature of the benzoate intermediate. observed for SOCL in comparison to DMAX demonstrates Theaddition of the chlorine substituent at the other ortho the advantage of our chemiluminescence probe over the position reduces the pKaofthe phenol and thus enriches the fluorescence-based approach. Theprobesselectivity towards 1 percentage of the phenoxy ion under physiological conditions. O2 among other ROSwas also evaluated. Remarkably,no Consequently,the chemiexcitation kinetics of the phenol- other ROScould produce light emission through the probe dioxetane is accelerated to enable the real-time monitoring of chemiexcitation pathway (Figure 2C). its formation. Next, we compared the activity of our SOCL probe with SOCL probe was synthesized according to apreviously that of methoxyvinylpyrene (MVP), achemiluminescence 1 [16] developed method (see the Supporting Information for probe for the detection of O2. This probe utilizes the [2+2] 1 synthetic details). Thechemiluminescence response of cycloaddition of O2 to an enol-ether to initiate achemilumi- SOCL to singlet oxygen was examined by incubation of the nescent decomposition process.MVP suffers from two major 1 probe with 3-(1,4-dihydro-1,4-epidioxy-4-methyl-1-naph- drawbacks.First, the reaction of MVP with O2 results in only thyl)propionic acid (EP-1). Thelatter is aknown water- 10%ofthe chemiluminescent dioxetane and 90%ofan 1 soluble compound that produces O2 through athermal undesired side product. Such circumstance reduces the decomposition.[15] As shown in Figure 2A, SOCL exhibited chemiluminescence quantum yield of the probe substan- tially.[17] In addition, MVP has ahighly hydrophobic structure that severely limits its water solubility,thus preventing the use of the probe at high concentrations under physiological conditions.These disadvantages become particularly signifi- 1 cant when trying to detect small amounts of O2,which is anyway difficult to detect in water owing to its short lifetime. Indeed, as shown in Figure 3, chemiluminescence measure-

Figure 2. A) Chemiluminescence kinetic profile of SOCL (500 mm) upon incubation with EP-1 (500 mm)inPBS, pH 7.4, 378C. B) Chem- iluminescence integrated signal upon incubation of SOCL (100 mm) with different concentrations of EP-1 for 30 min in PBS, pH 7.4, 378C. 1 C) Selectivity of SOCL (10 mm)towards O2 among other ROS (500 mm)inPBS, pH 7.4, 378C. Figure 3. Top: Integrated chemiluminescence signal of SOCL vs. MVP upon incubation with EP-1 (500 mm)for 3hin PBS, pH 7.4, 378C. A) Probe concentration (SOCL or MVP)=10 mm.B)Probe concentra- tion (SOCL or MVP)= 500 mm.Bottom:Chemical structures of SOCL an expected chemiluminescent exponential decay kinetic and MVP. profile in the presence of EP-1, while no chemiluminescence was obtained in the absence of EP-1 (for the chemilumines- cence spectrum see the Supporting Information, Figure S1). ments in the presence of EP-1 under physiological conditions ARP-HPLC assay showed complete conversion of SOCL to showed significant superiority of SOCL over MVP.Atlow 1 the dioxetane upon reaction with O2.The dioxetane decom- probe concentration (10 mm), SOCL generated about five poses to its corresponding benzoate ester,inaccordance with times more light than MVP,whereas at high probe concen- the mechanism presented in Figure 1(data are presented in tration (500 mm), the ratio between the signals produced by the Supporting Information, Figure S2). The SOCL probe was the probes is increased up to 100-fold. In addition, MVP emits

also incubated with different concentrations of EP-1. The light (lmax = 465 nm) while SOCL emits green light

obtained linear correlation between the EP-1 concentration (lmax = 515 nm). Green light is known to penetrate living and integrated chemiluminescence signal (Figure 2B)sug- tissue deeper than blue light.[18] Therefore, SOCL has agreater 1 1 gests that the O2 concentration could be straightforwardly potential to be further applied for in vivo O2 imaging than determined quantitatively.DMAX, aknown fluorescent MVP. 1 probe for O2, is considered by Nagano and co-workers as Photodynamic therapy (PDT) is gaining widespread 1 [7b] [19] 1 the “best fluorescence reagent for O2 detection”. SOCL clinical application as an anticancer treatment, and O2 is probe demonstrated extremely high sensitivity,with an LOD believed to be the primary cytotoxic agent that elicits cell 1 [4] for O2 below 500 nm,which is 10-fold lower than that of damage upon photosensitizer irradiation. Thus,itisessen- 1 DMAX (see the Supporting Information, Figure S3, for the tial to enable monitoring of intracellular O2 produced during

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PDT.Encouraged by the high sensitivity and selectivity of PDT.[21] Similarly to the SOCL-CPP probe,mTHPC also 1 observed for the SOCL probe towards O2,wesought to has aslight intrinsic fluorescence (see the Supporting assess whether our probe could be used for the detection of Information, Figure S5), which should be sufficient to detect 1 intracellular O2.Toincrease the cell permeability of the its ability for cell penetration. Figure 5B clearly shows that probe,wecovalently attached acell-penetrating peptide the mTHPC fluorescence signal appears inside the HeLa (CPP) to the acrylic acid moiety of SOCL.The synthesis of cells,demonstrating its cellular internalization. the modified probe was achieved as described in Figure 4. Once the cell internalization of SOCL-CPP and mTHPC was verified, we examined whether our probe is capable of 1 detecting intracellular O2 produced during the PDT process. HeLa cells were incubated with SOCL-CPP and mTHPC for 1h,and washed three times to remove any remaining probe or photosensitizer from the medium. Then, the cells were irradiated with light from awhite LED lamp for 60 sand their chemiluminescence signal was measured. Theexperiment was performed with two 96-well plates;one was kept in the dark as anegative control. As shown in Figure 6, astrong

Figure 4. Synthesis of SOCL-CPP,acell-permeable chemiluminescence singlet oxygen probe.

SOCL was reacted with N-hydroxy-succinimide (NHS) to form the corresponding NHS-ester 1.The latter was coupled with maleimide-amine 1a to generate maleimide derivative 1b.Reaction of nona-arginine–glycine–cysteine peptide (CPP,which has been shown to transport cargos into [20] cells) with the maleimide moiety of 1b afforded the Figure 6. A) Chemiluminescenceimagesobtained by HeLa cells SOCL-CPP probe. (2.5” 104/well) incubated with SOCL-CPP (10 mm), mTHPC(10 mm), or 1 In order to use SOCL-CPP as aprobe for O2 produced both for 1h.The bottom plate was irradiated with white LED lamp for inside the cells,wefirst verified its cell permeability. SOCL- 60 s, while the upper one was kept in the dark. B) Quantification of CPP also exhibits moderate fluorescent emission upon light intensities emitted from the cells. excitation at 405 nm (see the Supporting Information, Fig- ure S4). We used this intrinsic fluorescence to image SOCL- chemiluminescence signal was obtained by the cells incubated CPP cellular internalization by fluorescence confocal micros- with SOCL-CPP and mTHPC following light irradiation. copy.Asshown in Figure 5A,upon incubation of the SOCL- Conversely,nochemiluminescence signal was obtained from 1 CPP with human cervical adenocarcinoma HeLa cells,aclear cells that were incubated only with SOCL-CPP (no O2 1 intracellular fluorescence signal could be observed. This production) or only mTHPC (no O2 probe) following light observation indicates the internalization capability of SOCL- irradiation. No chemiluminescence signal was observed from CPP into the cells. the cells that were kept in the dark. 1 1 [22] To evaluate the adaptability of our probe to detect O2 in Addition of a O2-specific quencher (NaN3) results in PDT applications,wesought to determine whether the asignificant signal attenuation (see the Supporting Informa- internalization of an appropriate PDT photosensitizer could tion, Figure S6). PDT-induced cell death by mTHPC was also be imaged. Thephotosensitizer mTHPC (Foscan) has confirmed by standard cell-viability assay (see the Supporting been extensively investigated and used in clinical applications Information, Figure S7). This assay also demonstrated that SOCL-CPP by itself does not induce any light toxicity.This observation is especially significant because fluorescence- 1 based probes for O2 are known to suffer from their intrinsic ability to act as photosensitizers.[23] These results provide strong evidence for the ability of SOCL-CPP to detect 1 intracellular O2 produced during the PDT mode of action. It should be noted that the dioxetane intermediate formed by 1 the reaction of SOCL-CPP with O2 has ahalf-life of several minutes.This half-life is long enough to allow chemilumines- cence imaging of the PDT process,asdetection can be Figure 5. Imaging the cellular internalization of SOCL-CPP and performed shortly after light irradiation and 1O production. mTHPC in HeLa cells using confocalmicroscopy.A)HeLa cells were 2 In summary,wesynthesized and evaluated anew,efficient incubated with SOCL-CPP (10 mm)for 1hand washedthree times. B) HeLa cells were incubated with mTHPC (10 mm)and washed three chemiluminescence probe (SOCL), for the detection and 1 1 times. Excitation of SOCL-CPP and mTHPC at 405 nm. Scale imaging of O2.The probe reacts with O2 to form dioxetane, bar = 20 mm. which spontaneously decomposes under physiological con-

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ditions through achemiexcitation pathway to emit green light [4] M. T. Jarvi, M. J. Niedre,M.S.Patterson,B.C.Wilson, Photo- with extraordinary intensity. SOCL demonstrated high selec- chem. Photobiol. 2006, 82,1198 –1210. 1 [5] A. P. Losev,I.M.Byteva, G. P. Gurinovich, Chem. Phys.Lett. tivity and sensitivity towards O2.Acell-permeable version of 1988, 143,127 –129. the probe (SOCL-CPP)that includes nona-Arg as acell- [6] P. R. Ogilby, Chem. Soc.Rev. 2010, 39,3181 –3209. penetrating peptide was prepared and subjected to in vitro [7] a) N. Umezawa, K. Tanaka, Y. Urano,K.Kikuchi, T. Higuchi, T. studies. SOCL-CPP demonstrated apromising ability to Nagano, Angew.Chem. Int. Ed. 1999, 38,2899 –2901; Angew. 1 detect and image intracellular O2 produced by aphotosensi- Chem. 1999, 111,3076 –3079;b)K.Tanaka, T. Miura, N. tizer in HeLa cells during the PDT mode of action. In relation Umezawa,Y.Urano,K.Kikuchi, T. Higuchi, T. Nagano, J. to aqueous solubility and light emission intensity in water, our Am. Chem. Soc. 2001, 123,2530 –2536. probe exhibited significant advantages and superiority in [8] a) S. Kim, T. Tachikawa, M. Fujitsuka, T. Majima, J. Am. Chem. Soc. 2014, 136,11707 –11715;b)D.Song,S.Cho,Y.Han, Y. You, comparison to apreviously known chemiluminescence probe W. Nam, Org.Lett. , 15,3582 –3585;c)Z.Dai, L. Tian, Y. 1 2013 for O2.These findings make SOCL and SOCL-CPP as the Xiao,Z.Ye, R. Zhang,J.Yuan, J. Mater.Chem. B 2013, 1,924 – most effective known chemiluminescence probes for the 927;d)K.Xu, L. Wang,M.Qiang,L.Wang,P.Li, B. Tang, Chem. 1 detection of O2.Weanticipate that our chemiluminescence Commun. 2011, 47,7386 –7388;e)B.Song,G.Wang,M.Tan, J. 1 Yuan, J. Am. Chem. Soc. , 128,13442 –13450. probe for O2 imaging would be useful in PDT related 2006 applications,for example,for the evaluation of photosensi- [9] N. Hananya, A. Eldar Boock, C. R. Bauer,R.Satchi-Fainaro,D. tizers,and for the monitoring of 1O endogenously generated Shabat, J. Am. Chem. Soc. 2016, 138,13438 –13446. 2 [10] A. Gollmer,J.Arnbjerg,F.H.Blaikie,B.W.Pedersen, T. by cells in response to different stimuli. Breitenbach, K. Daasbjerg,M.Glasius,P.R.Ogilby, Photochem. Photobiol. 2011, 87,671 –679. [11] L. A. MacManus-Spencer,D.E.Latch, K. M. Kroncke,K. Acknowledgements McNeill, Anal. Chem. 2005, 77,1200 –1205. [12] A. P. Schaap,T.-S.Chen, R. S. Handley,R.DeSilva, B. P. Giri, D.S. thanks the Israel Science Foundation (ISF), the Bina- Tetrahedron Lett. 1987, 28,1155 –1158. [13] M. Matsumoto, J. Photochem. Photobiol. C 2004, 5,27–53. tional Science Foundation (BSF), and the German Israeli [14] O. Green, T. Eilon, N. Hananya, S. Gutkin, C. R. Bauer,D. Foundation (GIF) for financial support. This work is sup- Shabat, ACSCent. Sci. 2017, 3,349 –358. ported in part by agrant from the Israeli National Nano- [15] I. Saito,T.Matsuura, K. Inoue, J. Am. Chem. Soc. 1981, 103, technology Initiative (INNI), Focal Technology Area (FTA) 188 –190. program:Nanomedicine for Personalized Theranostics,and [16] G. H. Posner,J.R.Lever,K.Miura, C. Lisek, H. H. Seliger,A. by TheLeona M. and Harry B. Helmsley Nanotechnology Thompson, Biochem. Biophys.Res.Commun. 1984, 123,869 – Research Fund. R.S.-F.thanks the European Research 873. [17] A. Thompson, K. A. Canella, J. R. Lever,K.Miura, G. H. Council for the ERC Consolidator Grant Agreement n. Posner,H.H.Seliger, J. Am. Chem. Soc. 1986, 108,4498 –4504. [617445]- PolyDorm. [18] A. N. Bashkatov,E.A.Genina, V. I. Kochubey,V.V.Tuchin, J. Phys.D2005, 38,2543. [19] S. B. Brown, E. A. Brown, I. Walker, Lancet Oncol. 2004, 5,497 – Conflict of interest 508. [20] F. Madani,S.Lindberg, Ü.Langel, S. Futaki, A. Gräslund, J. Biophys. 2011 Theauthors declare no conflict of interest. 2011, ,414729. [21] M. O. Senge,J.C.Brandt, Photochem. Photobiol. 2011, 87, 1240 –1296. Keywords: chemiluminescence ·dioxetanes ·live-cell imaging · [22] M. Bancirova, 2011, 26,685 –688. molecular probes ·singlet oxygen [23] S. K. Pedersen, J. Holmehave,F.H.Blaikie,A.Gollmer,T. Breitenbach, H. H. Jensen, P. R. Ogilby, J. Org. Chem. 2014, 79, Howtocite: Angew.Chem. Int. Ed. 2017, 56,11793–11796 3079 –3087. Angew.Chem. 2017, 129,11955–11958

[1] B. C. Dickinson, C. J. Chang, Nat. Chem. Biol. 2011, 7,504 –511. [2] M. J. Davies, Biochem. Biophys.Res.Commun. 2003, 305,761 – Manuscript received:June 7, 2017 770. Revised manuscript received: July 24, 2017 [3] D. van Straten, V. Mashayekhi, H. de Bruijn, S. Oliveira, D. Acceptedmanuscript online: July 27, 2017 Robinson, Cancers 2017, 9,19. Version of record online: August 16, 2017

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