& Chemiluminescence | Hot Paper | Persistent Chemiluminescent Glow of Phenoxy-dioxetane Luminophore Enables Unique CRET-Based Detection of Proteases** Nir Hananya, [a] Ofir Press, [a] Alakesh Das, [b] Anna Scomparin, [c, d] Ronit Satchi-Fainaro, [c] Irit Sagi, [b] and Doron Shabat* [a] Abstract: Chemiluminescence is being considered an effec- tive imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to devel- opment of new phenoxy-dioxetane chemiluminescence lu- minophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a diox- etane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC- CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metallopro- teinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with signifi- cantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemilumi- nescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC- CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of pro- teins. Introduction Chemiluminescence is a powerful diagnostic tool for biosens- ing and bioimaging. [1–3] It offers considerable advantage over fluorescence in terms of signal-to-background ratio (S/B). Since in chemiluminescence imaging irradiation by an external light source is not required, interference by autofluorescence is cir- cumvented, and the obtained sensitivity is high. [4] Among known chemiluminescent compounds, the triggerable phen- oxy-dioxetanes are of the highest value for bioimaging. [5, 6] Light emission by these compounds is not activated by oxida- tion but rather by phenolate formation following deprotection of phenols. Therefore, by choice of an appropriate phenol-pro- tecting group, light emission can be associated with the activi- ty of a specific analyte. [7–9] A major breakthrough was recently achieved by our group, with the development of a new generation phenoxy-dioxetane luminophores. These bright luminophores emit light under physiological conditions with very high efficiency. [10, 11] We and others have utilized these dioxetane luminophores to con- struct chemiluminescent probes for the detection and imaging of various enzymes and chemical analytes. [12–14] Such probes are useful for non-invasive chemiluminescence imaging of living cells and in animals. [15–19] Despite this considerable progress, the phenoxy-dioxetane probes suffer from an inherent restriction: They can be turned- on only by phenol deprotection. For example, in a classic diox- etane-based probe for a protease, the phenol group of the lu- minophore is masked with a peptide substrate through a 4- aminobenzyl alcohol self-immolative linker (Figure 1 A). Sub- strate recognition and hydrolysis by the protease of interest [a] Dr. N. Hananya, O. Press, Prof. D. Shabat School of Chemistry Raymond and Beverly Sackler Faculty of Exact Sciences Tel Aviv University, Tel Aviv 6997801 (Israel) E-mail : chdoron@post.tau.ac.il [b] Dr. A. Das, Prof. I. Sagi Department of Biological Regulation Weizmann Institute of Science, Rehovot 7610001 (Israel) [c] Dr. A. Scomparin, Prof. R. Satchi-Fainaro Department of Physiology and Pharmacology, Sackler Faculty of Medicine Tel Aviv University, Tel Aviv 6997801 (Israel) [d] Dr. A. Scomparin Department of Drug Science and Technology University of Turin, Via P. Giuria 9, 10125 Turin (Italy) [**] CRET = chemiluminescence resonance energy transfer. Supporting Information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/chem.201903489. Chem. Eur. J. 2019, 25, 1 – 10 # 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 && These are not the final page numbers! ÞÞ Full Paper DOI: 10.1002/chem.201903489