Designing ¹²⁹Xe NMR Biosensors for Matrix Metalloproteinase Detection

Abstract

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 microM, V(max) = 1.3 x 10(-)(8) M s(-1), k(cat)/K(M) = 7,200 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 +/- 1,000 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.

Measurement of radon and xenon binding to a cryptophane molecular host

“…Thus, the design of higher affinity xenon-binding molecules such as TTEC extends the range of possible biological applications. Biologically targeted cryptophanes are under development as potential 129 Xe MRI contrast agents, as demonstrated by xenon biosensors for the prototypical biotin-avidin interaction (53,54) and other proteins (55)(56)(57), as well as preliminary cell studies (58)(59)(60). This demonstration of radon binding to cryptophane raises similar possibilities of molecularly functionalized radon binders for biological, environmental, and materials applications involving radon delivery, sequestration, or detection.…”

Development of an antibody-based, modular biosensor for129Xe NMR molecular imaging of cells at nanomolar concentrations

“…Xenon can be hyperpolarized, enhancing its signal by up to five orders of magnitude and enabling it to be detected at extremely low concentrations (5). In addition, the exquisite sensitivity of xenon to its local chemical environment, as indicated by wellresolved chemical shift changes, brings with it the potential to distinguish not only localization but also specific functional events (6)(7)(8)(9)(10). This sensitivity may also be exploited in the development of different xenon MRI contrast agents which could be used to perform multiplexed molecular imaging (11), especially useful as clusters of biomarkers are often more informative than a single indicator.…”

129Xenon NMR: Review of recent insights into porous materials

“…Xenon encapsulated into cryptophane derivative cages can be detected and exploited by NMR spectroscopy. The great sensitivity of xenon to local environment combined with the use of hyperpolarization techniques led to an important variation of the NMR chemical shifts [231][232][233]. Additional signal amplification can be obtained by chemical exchange saturation transfer (hyperCEST) [234,235].…”