Design and synthesis of targeted cryptophane biosensors for xenon-129 NMR/MRI

To expand the utility of hyperpolarized (hp) 129Xe NMR for sensitive biodetection, a cryptophane host molecule can be specifically targeted to analytes of interest. This system has the potential to be used in conjunction with proton MRI for the diagnosis and staging of disease. Xenon was found previously to have high affinity (KD = 20-30 µM at room temperature) for trifunctionalized, water-soluble cryptophanes. Cryptophanes have been designed for increased water solubility and xenon affinity, and provide a broad (~300 ppm) chemical shift window. Importantly, new synthetic methods have enabled conjugation of a variety of targeting and solubilizing ligands, and increased access to enantiopure cryptophane. This dissertation reports my progress in three areas. The first involves studies with carbonic anhydrase, a useful model system for the design and characterization of xenon biosensors targeted to enzymes indicated in disease progression. Cryptophane functionalized with a benzenesulfonamide ligand and two water-solubilizing moieties bound to carbonic anhydrase II (CAII) with nanomolar affinity; Zn2+ coordination in the active-site channel was confirmed by X-ray crystallography. Using xenon biosensors tailored for CA, progress has been made in manipulating and better resolving the 129Xe NMR chemical shift, as required for multiplexing studies. The second study details the development of a biosensor that labels cells in an acidic microenvironment. Cryptophane was conjugated to a peptide that undergoes a conformational change from random coil to alpha-helical as the pH decreases from 7.5 to 5.5. This conformational change stimulates membrane insertion, as validated with HeLa cell studies. With this compound we demonstrated the engineering of the largest published chemical shift change for cryptophane biosensor. We also utilized hp 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR methods to achieve low-picomolar cryptophane detection, which creates new opportunities for bio-analysis and molecular imaging. Finally, a peptido-cryptophane was developed that binds to calmodulin (CaM) protein. With this work we hope to not only demonstrate the development of a biophysical probe sensitive to changes in protein conformation but also generate a Ca2+ sensor. These three studies highlight the broad utility of cryptophane-129Xe NMR biosensors, and synthetic methods described herein lay the groundwork for future in vivo studies