Tissue oxygenation is a driving parameter of the tumor microenvironment and hypoxia can be a prognostic indicator of aggressiveness, metastasis, and poor response to therapy. Here we report a chemiluminescence imaging (CLI) agent based on the oxygen-dependent reduction of a nitroaromatic spiroadamantane 1,2-dioxetane scaffold. Hypoxia ChemiLuminescent Probe 2 (HyCL-2) responds to nitroreductase with ~170-fold increase in luminescence intensity, with high selectivity for enzymatic reductase versus other small molecule reductants. HyCL-2 can image exogenous nitroreductase in vitro and in vivo in living mice and total luminescent intensity is increased by ~5-fold under low oxygen conditions. HyCL-2 is demonstrated to report on tumor oxygenation during oxygen challenge in H1299 lung tumor xenografts grown in a murine model as independently confirmed using multi-spectral optoacoustic tomography (MSOT) imaging of hemoglobin oxygenation.

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