Designing 129Xe NMR biosensors for matrix metalloproteinase detection

Xenon-129 biosensors offer an attractive alternative to conventional MRI contrast agents due to the chemical shift sensitivity and large nuclear magnetic signal of hyperpolarized Xe-129. Here, we report the first enzyme-responsive Xe-129 NMR biosensor. This compound was synthesized in 13 steps by attaching the consensus peptide substrate for matrix metalloproteinase-7 (MMP-7), an enzyme that is upregulated in many cancers, to the xenon-binding organic cage, cryptophane-A. The final coupling step was achieved on solid support in 80-92% yield via a copper (I)-catalyzed [3+2] cycloaddition. In vitro enzymatic cleavage assays were monitored by HPLC and fluorescence spectroscopy. The biosensor was determined to be an excellent substrate for MMP-7 (K-M = 43 mu M, V-max = 1.3 x 10(-8) M s(-1), k(cat)/K-M = 7200 M-1 s(-1)). Enzymatic cleavage of the tryptophan-containing peptide led to a dramatic decrease in Trp fluorescence, lambda(max) = 358 nm. Stern-Volmer analysis gave an association constant of 9000 +/- 1000 M-1 at 298 K between the cage and Trp-containing hexapeptide under enzymatic assay conditions. Most promisingly, Xe-129 NMR spectroscopy distinguished between the intact and cleaved biosensors with a 0.5 ppm difference in chemical shift. This difference most likely reflected a change in the electrostatic environment of Xe-129, caused by the cleavage of three positively charged residues from the C-terminus. This work provides guidelines for the design and application of new enzyme-responsive Xe-129 NMR biosensors.