A recent methodology, developed by our group, has enabled a dramatic improvement in the emissive nature of the excited species, formed during the chemiexcitation of dioxetanes under physiological conditions. This approach has resulted in the discovery of distinct phenoxy-dioxetane luminophores that produce a chemiluminescence signal via a direct-mode of emission. Here, we show a significant pK effect of our new phenoxy-dioxetanes on their chemiexcitation and on their ability to serve as chemiluminescent turn-ON probes for biological applications. Using an appropriate phenoxy-dioxetane probe with a direct-mode of emission, we were able to image β-galactosidase activity, in cancer cells and in tumor-bearing mice. To the best of our knowledge, this is the first example to demonstrate in vitro and in vivo endogenous enzymatic chemiluminescence images obtained by a single-component phenoxy-dioxetane probe. We anticipate that our strategy, for the design and synthesis of such distinct luminophores, will assist in providing new effective turn-ON probes for non-invasive intravital chemiluminescence imaging techniques.

Understanding the correlation between structural features of small-molecule drugs and their mode of action is a fascinating topic and crucial for the drug-discovery process. However, in many cases, knowledge of the exact parameters that dictate the mode of action is still lacking. Following a large screening for ubiquitin specific protease 2 (USP2) inhibition, an effective para-quinone-based inhibitor with an unclear mode of action was identified. To gain a deeper understanding of the mechanism of inhibition, a set of para-quinones were prepared and studied for USP2 inhibition, electrocatalysis, and reactive oxygen species (ROS) quantification. The excellent correlation obtained from the above-mentioned studies disclosed a distinct pattern of "N-C=O-N" in the bicyclic para-quinones to be a crucial factor for ROS generation, and demonstrated that minor changes in such a skeleton drastically altered the ROS-generating ability. The knowledge acquired herein would serve as an important guideline for future medicinal chemistry optimization of related structures to select the preferred mode of action.

The cover picture shows that bicyclic para‐quinones with a distinct “N‐C=O‐N” pattern are able to generate reactive oxygen species (ROS) catalyzing the reduction of oxygen, although minor changes in such a skeleton drastically alter its ROS‐generating ability. In their article, the authors provide a structure–activity relationship study of these para‐quinones and discuss their ability to inhibit ubiquitin‐specific protease 2 through ROS production. The substantiation of the mode of action of these inhibitors is supported by cyclic voltammetry and hydrogen peroxide quantification studies. The knowledge acquired in this study should serve as an important guideline for future medicinal chemistry optimization of related structures so as to select the preferred mode of action of para‐quinone‐based inhibitors. More information can be found in the communication by D. Shabat, Z. Gross, A. Brik, et al. on page 1683 in Issue 17, 2017 (DOI: 10.1002/cbic.201700330).