Designing ~(129)Xe NMR Biosensors for Matrix Metalloproteinase Detection

摘要

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 ~(129)Xe.Here,we report the first enzyme-responsive ~(129)Xe 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,~(129)Xe 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 ~(129)Xe,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 ~(129)Xe NMR biosensors.