Enantiopure Cryptophane-¹²⁹Xe Nuclear Magnetic Resonance Biosensors Targeting Carbonic Anhydrase

The (+) and (-) enantiomers for a cryptophane-7-bond-linker-benzenesulfonamide biosensor (C7B) were synthesized and their chirality confirmed by electronic circular dichroism (ECD) spectroscopy. Biosensor binding to carbonic anhydrase II (CAII) was characterized for both enantiomers by hyperpolarized (hp) ¹²⁹Xe NMR spectroscopy. Our previous study of the racemic (+/-) C7B biosensor-CAII complex [Chambers, et al., J. Am. Chem. Soc. 2009, 131, 563-569], identified two "bound" ¹²⁹Xe@C7B peaks by hp ¹²⁹Xe NMR (at 71 and 67 ppm, relative to "free" biosensor at 64 ppm), which led to the initial hypothesis that (+) and (-) enantiomers produce diastereomeric peaks when coordinated to Zn²⁺ at the chiral CAII active site. Unexpectedly, the single enantiomers complexed with CAII also identified two "bound" ¹²⁹Xe@C7B peaks: (+) 72, 68 ppm and (-) 68, 67 ppm. These results are consistent with X-ray crystallographic evidence for benzenesulfonamide inhibitors occupying a second site near the CAII surface. As illustrated by our studies of this model protein-ligand interaction, hp ¹²⁹Xe NMR spectroscopy can be useful for identifying supramolecular assemblies in solution.