1,2,4,5-Tetrazine-tethered probes for fluorogenically imaging superoxide in live cells with ultrahigh specificity

Specific detection of cellular superoxide is challenging. Here, the authors designed 1,2,4,5-tetrazine based fluorogenic probes for specific and sensitive imaging of superoxide, and applied them in high throughput screening of modulators of oxidative stress.

Redox biology is intriguing. It is vital for a variety of normal cell functions, including growth, survival, and proliferation. On the other hand, abnormal redox biology, usually in the form of oxidative stress (OS), causes diseases. A large body of evidence has implicated OS in the pathology of neurodegenerative and metabolic diseases. However, the continuous clinical failure of small-molecule-antioxidants keeps reminding people the complexicity of redox biology, and the necessity to recognize redox biology with more accuracy. Reactive oxygen species (ROS) are the primary players in redox biology. Howerver, “ROS” cover a variety of species with distinct reactivity and spatiotemporal distribution profiles. To study redox biology with precision, it is prerequisite to recognize the specific ROS with specificity.

We are especially interested in the specific recognition of superoxide which is the primary ROS specie in redox biology. Superoxide in produced by mitochondrial electron transport chain, NADPH oxidases, and other cellular oxidases. It is readily transformed to hydrogen peroxide by superoxide dismutases. It can also react with nitric oxide in a diffusion-controlled rate to form peroxynitrite, which is highly oxidative and destructive. We think superoxide is important for both oxidative eustress and distress. Although there are some assays available for superoxide detection, such as EPR, HPLC, chemiluminescence, and fluorescence imaging, etc, all are far from being ideal. In this context, we are especially interested in an assay that is capable of detecting superoxide in live cells with high selectivity and spatiotemporal resolution.

The most interesting reactivity of superoxide is its reducibility. For example, superoxide is capable of donating one electron to nitro blue tetrazolium, by which a chromogenic assay is developed for detecting superoxide. Inspired by this unique reactivity, we calculated the electron-accepting ability of a variety of N-containing heterocyclic compounds, and found that 1,2,4,5-tetrazine has an electron affinity slightly higher than the ionization potential of superoxide, indicating its potential to be reduced by superoxide. Setting out from this point, we developed a series of tetrazine-based probes for sensing or imaging superoxide. Much to our delight, these probes demonstrated a high degree of selectivity towards superoxide among the various oxidative species. Some even outperformed MitoSOX (a traditional superoxide probe) to a much high extent in cell imaging experiments.

We think we are kind of being lucky to find the tetrazine moiety as a superoxide sensing trigger, as this moiety has been well recognized for its fluorescence quenching ability. This feature facilitates the probe design procedures, and fluorogenic probes for superoxide can be modularly designed simply by tethering the tetrazine moiety into various fluorophores. In this way, probes with rainbow-like emission colors can be straightforwardly designed.

With the above exciting results, we decided to leverage the probes to develop a high-content screening model for superoxide modulators to conteract its over-accumulation. We used probe F-Tz4 for this purpose. Thanks to its ultrafluorogenic response, the screening model could be estsblished with a high degree of sensitivity, and we were able to identify several hits from a house-hold natural product library. Among the hit compounds, coprostanone which is a microbial metabolite of cholesterol, aroused our interest, as this antioxidant activity is new for it. We then constructed a mice model of myocardial ischemia/reperfusion injury to test this antioxidant activity of coprostanone, and confirmed its myocardial protective effect by inhibiting superoxide overload.

In summary, we provided a series of tetrazine-based fluorogenic probes for imaging superoxide in live cells. We envision the application of these probes to accelerate the precision study on redox biology.

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