Designing 129Xe NMR biosensors for matrix metalloproteinase detection
Designing 129Xe NMR biosensors for matrix metalloproteinase detection
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DOI：
10.1021/ja0640501
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发表时间：
2006-10-11
影响因子：
15
通讯作者：
Dmochowski, Ivan J.
中科院分区：
文献类型：
--
作者：
Wei, Qian;Seward, Garry K.;Dmochowski, Ivan J.
由于超极化 Xe-129 的化学位移敏感性和大核磁信号，Xenon-129 生物传感器为传统 MRI 造影剂提供了一种有吸引力的替代品。在这里，我们报告了第一个酶响应 Xe-129 NMR 生物传感器。该化合物是通过将基质金属蛋白酶 7 (MMP-7)（一种在许多癌症中上调的酶）的共有肽底物连接到氙结合有机笼 Cryptophane-A 上，通过 13 个步骤合成的。最终的偶联步骤通过铜 (I) 催化的 [3+2] 环加成在固体载体上实现，产率为 80-92%。通过 HPLC 和荧光光谱监测体外酶裂解测定。该生物传感器被确定为MMP-7的优良底物（K-M = 43 mu M，V-max = 1.3 x 10(-8) M s(-1)，k(cat)/K-M = 7200 M-1 s(-1)）。含色氨酸肽的酶裂解导致色氨酸荧光急剧下降，λ(max) = 358 nm。 Stern-Volmer 分析表明，在酶测定条件下，笼与含色氨酸六肽之间在 298 K 下的缔合常数为 9000 +/- 1000 M-1。最有希望的是，Xe-129 NMR 光谱法可以区分完整和裂解的生物传感器，其化学位移差异为 0.5 ppm。这种差异很可能反映了 Xe-129 静电环境的变化，这是由 C 末端三个带正电的残基裂解引起的。这项工作为新型酶响应 Xe-129 NMR 生物传感器的设计和应用提供了指导。
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.