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

We report the microwave-assisted synthesis of a novel family of peptide-linked optical imaging probes incorporating the <i>L</i>-[7,13] bombesin fragment (denoted <i>L</i>-[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 <i>L</i>- and <i>D</i>-amino acid-substituted naphthalenediimide (NDI), namely, the monopeptide (<i>L</i>-<b>3</b>) and corresponding bis-peptide (<i>L</i>-<b>4</b>) 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 (<i>L</i>-<b>6</b>), which incorporates the <i>L</i>-[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 <i>L</i>- and <i>D</i>-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 <i>L</i>-<b>6</b> 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.