Click Bioconjugation of Aza‐BODIPY Fluorophores to LfcinB Peptides for Improved Biocompatibility

Near‐infrared (NIR) organic fluorophores have emerged as promising for biomedical imaging and theragnostics, offering deep‐tissue penetration and reduced background interference. However, poor aqueous solubility and limitations in photostability, biocompatibility, and targeting hinder in vivo applications. Similarly, antimicrobial and anticancer peptides exhibit proteolytic instability and rapid clearance. This study addresses these issues by covalently bioconjugating aza‐BODIPY fluorophores with LfcinB‐derived peptides (RRWQWR, RWQWRWQWR) using CuAAC and thiol‐maleimide click chemistries. Two strategies were compared: (i) CuAAC between azide/alkyne‐functionalized partners, and (ii) Michael addition with cysteine‐containing peptides and AZB‐maleimide . Bioconjugation significantly improved water solubility (calculated LogP 5.3–5.5 → 1.1–4.2; LogS −8.2 → −2.2/−3.7) while preserving NIR photophysical properties ( λ ab s = 703–730 nm). In silico ADMET analysis provided preliminary support for favorable predicted absorption‐related properties and metabolic stability. Thiol‐maleimide constructs showed superior predicted solubility, lower mutagenicity, and more favorable synthetic accessibility than CuAAC analogs. Phase‐contrast microscopy of HeLa cells revealed characteristic apoptotic morphology (cell rounding/shrinkage, membrane association) with blue staining reflecting aza‐BODIPY localization. In parallel, P12 induced caspase activation in flow cytometry assays, providing preliminary evidence of caspase‐associated apoptotic activity. Collectively, these platforms provide a basis for the further development of NIR probes, although their in vivo performance remains to be established experimentally.