Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

中文翻译：

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19F取代的氨基酸，作为荧光团标记的替代物：通过19F NMR监测渗透和类似物Penetratin类似物的降解。

当前评估细胞穿透肽（CPPs）的细胞摄取的方法通常依赖于荧光团标记的CPP的检测。然而，荧光探针的引入通常赋予改变的理化性质，使得荧光团-CPP缀合物可以表现出天然CPP没有施加的细胞毒性作用和膜损伤。在本研究中，对氟探针的引入进行了研究，以替代CPP的荧光团标记，因为这只会对其总体理化特性产生微小的影响。19F NMR光谱的高灵敏度以及天然存在的氟的背景信号的缺失使得能够检测内在化的CPP。同样，在暴露于Caco-2细胞的过程中，氟标记的肽的降解可以通过使用19F NMR光谱进行跟踪。总共，通过使用可商购的氟化氨基酸作为标记，合成了五种CPP渗透肽模型的氟化类似物，包括一个也带有N末端荧光团的类似物。荧光团-渗透素缀合物的表观细胞摄取明显更高，表明荧光团部分促进了肽的摄取。19F NMR光谱的使用可通过建立摩尔平衡并通过摄取后验证CPP完整性来监测CPP的命运。因此，与目前依赖荧光检测的广泛使用的方法相比，基于NMR的方法具有多个优点。本研究结果为改进CPPs的标记策略提供了指导，从而扩大了可用于研究CPPs降解和吸收的分析技术的全部范围。