Persistent Chemiluminescent Glow of Phenoxy‐dioxetane Luminophore Enables Unique CRET‐Based Detection of Proteases

Abstract

Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy‐dioxetane chemiluminescence luminophores, 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 dioxetane 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 metalloproteinases, 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 significantly higher signal‐to‐background ratio than an analogous fluorescence resonance energy transfer (FRET)‐based probe. This work is expected to open new horizons in chemiluminescence 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 proteins.

Prevent or Cure—The Unprecedented Need for Self‐Reporting Materials

“…[123,125,132] By carefully choosing the phenol substituents (and their position) of Schaapsd ioxetane discovered in 1987, aplethora of CL 1,2-dioxetane derivatives for (bio-)labeling and imaging have been developed, which are also suitable for in vivo applications under physiological conditions. [126,[171][172][173][174][175][176][177] Besides the thermal and chemical activation, the CL of 1,2-dioxetanes can also be triggered by mechanical forces. Theincorporation of bis(adamantyl)1,2-dioxetanes into polymeric materials (e.g.p olyurethane, [178][179][180][181] poly(methyl methacrylate), [182] poly(methyl acrylate), [183,184] )p oly(dimethylsiloxane) [185] )e nabled the facile,r eal-time monitoring of bond breaking events in such materials,s ince mechanical force leads to ab ond scission of the dioxetane moiety into two adamantone-terminated polymer chain visualized by the emission of light, as can be seen in Scheme 3C.C learly,1 ,2dioxetanes represent an important class for the development of versatile,s mart, self-reporting materials since the CL can be easily tailored to the desired stimulus by thorough choice of the respective 1,2-dioxetane derivative.…”

“…[ 123 , 125 , 132 ] By carefully choosing the phenol substituents (and their position) of Schaap's dioxetane discovered in 1987, a plethora of CL 1,2‐dioxetane derivatives for (bio‐)labeling and imaging have been developed, which are also suitable for in vivo applications under physiological conditions. [ 126 , 171 , 172 , 173 , 174 , 175 , 176 , 177 ]…”

Vorbeugen oder Heilen – die beispiellose Notwendigkeit von selbstberichtenden Materialien

“…[123,125,132] Durch eine wohlüberlegte Auswahl der Phenol-Substituenten (und ihrer Position) des 1987 entdeckten Schaap-Dioxetans wurde eine Fülle an CL-1,2-Dioxetan-Derivaten zur (Bio-)Markierung und Bildgebung entwickelt, die auch fürI n-vivo-Anwendungen unter physiologischen Bedingungen geeignet sind. [126,[171][172][173][174][175][176][177] Neben der thermischen und chemischen Aktivierung kann die CL von 1,2-Dioxetanen auch durch mechanische Kräfte ausgelçst werden. Der Einbau von Bis(adamantly)-1,2-dioxetanen in polymere Materialien (z.…”

“…Während dieses Prozesses zersetzt sich das entschützte Dioxetan und es entsteht eine angeregte Benzoatester‐Spezies, die unter Lichtemission in den Grundzustand zurückkehrt (Schema 3 B). [123, 125, 132] Durch eine wohlüberlegte Auswahl der Phenol‐Substituenten (und ihrer Position) des 1987 entdeckten Schaap‐Dioxetans wurde eine Fülle an CL‐1,2‐Dioxetan‐Derivaten zur (Bio‐)Markierung und Bildgebung entwickelt, die auch für In‐vivo‐Anwendungen unter physiologischen Bedingungen geeignet sind [126, 171–177] …”

Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

“…69 FRET also allows for fluorophores to be paired to dyes with sensing capabilities, thus providing ratiometric detection of analytes with the FRET pair. [70][71][72][73] It is currently known that 1,2-dioxetanes exhibit energy transfer capabilities, [74][75][76] but to our knowledge, no single-molecule ratiometric chemiluminescent probes for imaging pH have been developed. Here, we report the first single molecule ratiometric chemiluminescence resonance energy transfer (CRET) sensor quantifying pH in living systems (Scheme 1).…”