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 (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.
Wei
Qian
Q
Department of Chemistry, University of Pennsylvania, USA.
Seward
Garry K
GK
Hill
P Aru
PA
Patton
Brian
B
Dimitrov
Ivan E
IE
Kuzma
Nicholas N
NN
Dmochowski
Ivan J
IJ
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D007700:Kinetics; D020783:Matrix Metalloproteinase 7 / Q000032:analysis* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D013050:Spectrometry, Fluorescence; D014979:Xenon Isotopes / Q000737:chemistry*
D014979:Xenon Isotopes; D020783:Matrix Metalloproteinase 7