Abstract: | The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in 1H MRI are impaired by the high abundance of protons in the body. 129Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional 1H MRI because of its ability to report specific chemical environments. To date, the scope of responsive 129Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane‐based 129Xe hyperCEST NMR contrast agent that can be turned on in response to H2O2, which is upregulated in several disease states. Added H2O2 was detected by 129Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H2O2 produced by HEK 293T cells activated with tumor necrosis factor. |