Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors,Analytical Chemistry

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Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-04-14 , DOI: 10.1021/acs.analchem.1c00280
Yongchao Liu ₁ , Chengyan Xu ₁ , Hong-Wen Liu _{1,

2} , Lili Teng ₁ , Shuangyan Huan ₁ , Lin Yuan ₁ , Xiao-Bing Zhang ₁

Affiliation

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 in situ 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 de novo precipitated fluorophore, HBPQ, 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 HBPQ by the protection–deprotection of phenolic hydroxyl, we further utilized HBPQ to design an APN-responsive fluorogenic probe (HBPQ-A) for the imaging of intracellular APN. Importantly, HBPQ-A can not only perform in situ imaging of APN in different organelles (e.g., 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 HBPQ-A 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 in situ imaging.

中文翻译：

基于荧光团的沉淀分子探针，用于活细胞和肿瘤中氨肽酶N的原位成像

氨基肽酶N（APN）能够从位于N末端位置有丙氨酸的肽中裂解N末端氨基酸，并在癌症的生长，迁移和转移中起关键作用。但是，当前的APN反应性分子探针难以获得可靠的原位信息，因为释放的荧光团是可溶于细胞质的，因此会迅速脱离酶促反应位点并散布到整个细胞质中。在这里，我们报道了从头开始沉淀的荧光团HBPQ，它完全不溶于水，并在用405 nm激光激发时显示出强烈的黄色固体发射。由于HBPQ的可控固体荧光通过酚羟基的保护-脱保护，我们进一步利用HBPQ设计了APN响应型荧光探针（HBPQ-A），用于细胞内APN的成像。重要的是，HBPQ-A不仅可以在不同细胞器（例如，溶酶体，线粒体，内质网等）中对APN进行原位成像，而且还可以显示稳定且不可扩散的荧光信号，以可靠地绘制生活中APN的分布图细胞。此外，通过对4T1肿瘤中APN的实时成像，我们发现HBPQ-A产生的高保真荧光信号甚至在12 h后仍能保持恒定，这进一步证实了其扩散抗性和长期可靠的成像能力。我们相信沉淀的荧光团可能具有长期原位成像的巨大潜力。

更新日期：2021-04-28

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