Functional D - and L -Naphthalenediimide-Peptides: Microwave-Driven Synthesis, Supramolecular Aggregation, and Multiphoton Fluorescence Lifetime Imaging Microscopy in Living Cells

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High Resolution Image Download MS PowerPoint Slide We report the microwave-assisted synthesis of a novel family of peptide-linked optical imaging probes incorporating the L -[7,13] bombesin fragment (denoted L -[7,13]BBN) as a functional building block currently used for targeting the gastrin-releasing peptide receptor (GRPR) in cancer cells. Given the importance of chirality in probe design, we synthesized and evaluated both L - and D -amino acid-substituted naphthalenediimide (NDI), namely, the monopeptide ( L - 3 ) and corresponding bis-peptide ( L - 4 ) conjugates. These bioconjugates were characterized using NMR, fluorescence spectroscopy, including excitation–emission mapping, and mass spectrometry, confirming their spectroscopic tunability, water solubility, and ability to form supramolecular aggregates. Aggregation behavior was demonstrated by scanning electron microscopy (SEM) and Time-Correlated Single-Photon Counting (TCSPC) spectroscopy, while circular dichroism studies revealed a stereochemistry-driven self-assembly influenced by 4-iodophenylalanine modifications. Additionally, a new, desymmetrized NDI-based bioconjugate ( L - 6 ), which incorporates the L -[7,13]BBN fragment and a functional BODIPY fluorescent label, was synthesized in a stepwise manner via the microwave-assisted methods developed hereby. Cytotoxicity assays showed that these are benign, nontoxic probes at the time of imaging experiments and up to 72 h observation. Cellular uptake and localization properties of all compounds were assessed using confocal laser-scanning microscopy correlated with multiphoton fluorescence lifetime imaging microscopy (MP FLIM). This imaging method provided insights into the distinct behaviors of mono- vs bis-substituted peptide conjugates in live PC-3 prostate cancer cells, known to overexpress GRPR, and in A431 cells, known to overexpress the epidermal growth factor receptor (EGFR). Notably, the L - and D -stereochemistries of the BBN[7,13] fragment played a crucial role in modulating the uptake and subcellular localization of bioconjugates of type 3 and 4 in lysosomes while the presence of the BODIPY unit additionally directed the biolocalization of compound L - 6 toward the endoplasmic reticulum of multiple cellular environments, including in living PC-3 and A431 cells. These findings are relevant for the design of new biologically active probes, including proteolysis-inactive, peptide conjugates for cancer biomarker detection and imaging.

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