School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, 7610001, Israel.
Chemistry. 2019 Nov 18;25(64):14679-14687. doi: 10.1002/chem.201903489. Epub 2019 Oct 18.
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.
化学发光被认为是一种有效的成像方式，因为它具有低背景和高灵敏度。我们小组的最近发现导致了新的苯氧基-二氧杂环丁烷化学发光发光体的开发，这些发光体在生理条件下高度明亮。然而，基于这些发光体的探针目前的应用范围有限，因为它们只能通过酚保护基团的去除来打开。在这里，我们提出了一种新的化学发光共振能量转移（CRET）系统，Glow-CRET，其中光发射是通过连接二氧杂环丁烷发光体和猝灭剂的肽底物的蛋白水解切割触发的。为了组成这样的系统，开发了一种新的苯氧基-二氧杂环丁烷发光体 7-HC-CL。这种发光体表现出强烈和持久的发光化学发光；它经历非常缓慢的化学激发，并且在生理条件下具有迄今为止报道的最高化学发光量子产率。基于 7-HC-CL，合成了基质金属蛋白酶的 Glow-CRET 探针 MMP-CL。MMP-CL 与其同源蛋白酶孵育导致化学发光信号增加 160 倍。MMP-CL 还能够以比类似的荧光共振能量转移（FRET）探针更高的信号背景比检测癌细胞中的基质金属蛋白酶活性。这项工作有望在化学发光成像方面开辟新的视野，因为它能够以以前不可能的方式使用二氧杂环丁烷。我们预计 7-HC-CL 和未来的衍生物不仅将用于构建进一步的 Glow-CRET 探针，还将用于其他应用，例如蛋白质的化学发光标记。
