State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410082, P. R. China.
Anal Chem. 2021 Apr 27;93(16):6463-6471. doi: 10.1021/acs.analchem.1c00280. Epub 2021 Apr 14.
Aminopeptidase N (APN) is capable of cleaving N-terminal amino acids from peptides with alanine in the N-terminal position and plays a key role in the growth, migration, and metastasis of cancer. However, reliable information is hard to be obtained with the current APN-responsive molecular probes because the released fluorophores are cytoplasmic soluble and thus rapidly depart from the enzymatic reaction sites and spread out all over the cytoplasm. Here, we report a precipitated fluorophore, , which is completely insoluble in water and shows strong yellow solid emission when excited with a 405 nm laser. Owing to the controllable solid fluorescence of by the protection-deprotection of phenolic hydroxyl, we further utilized to design an APN-responsive fluorogenic probe () for the imaging of intracellular APN. Importantly, can not only perform imaging of APN in different organelles (, lysosomes, mitochondria, endoplasmic reticula, and so forth) but also display a stable and indiffusible fluorescent signal for reliable mapping of the distribution of APN in living cells. In addition, through real-time imaging of APN in 4T1 tumors, we found that the fluorescent signal with high fidelity generated by could remain constant even after 12 h, which further confirmed its diffusion-resistant ability and long-term reliable imaging ability. We believe that the precipitated fluorophore may have great potential for long-term imaging.
天冬氨酰蛋白酶 N（APN）能够从 N 末端为丙氨酸的肽段中切割 N 末端氨基酸，在癌症的生长、迁移和转移中发挥关键作用。然而，由于当前的 APN 响应性分子探针释放的荧光团是细胞质可溶性的，因此它们会迅速离开酶反应位点并扩散到整个细胞质中，因此很难获得可靠的信息。在这里，我们报告了一种沉淀荧光团 ，它在水中完全不溶，当用 405nm 激光激发时显示出强黄色固体发射。由于酚羟基的保护-脱保护可控制 的固态荧光，我们进一步利用 设计了一种用于细胞内 APN 成像的 APN 响应性荧光探针（）。重要的是，不仅可以对不同细胞器（、溶酶体、线粒体、内质网等）中的 APN 进行成像，而且可以显示出稳定且不可扩散的荧光信号，从而可靠地定位活细胞中 APN 的分布。此外，通过对 4T1 肿瘤中 APN 的实时成像，我们发现 产生的具有高保真度的荧光信号即使在 12 小时后仍保持不变，这进一步证实了其抗扩散能力和长期可靠成像能力。我们相信沉淀荧光团在长期成像方面可能具有巨大的潜力。
