Minimal-tag probes for intracellular imaging of polyamines

Polyamines (putrescine, spermidine, and spermine) are essential cellular polycations that regulate chromatin structure, RNA function, and organelle physiology. However, the lack of minimally perturbing probes has limited visualization of their intracellular dynamics. Here, we report the design, synthesis, and application of propargyl analogs of polyamines (iPUT, iSPD, and iSPM) as minimal-tag, bioorthogonal probes for high-resolution imaging in mammalian cells and zebrafish. These probes preserve the physicochemical features and transport compatibility of native polyamines, enabling fixation-based labeling via copper-catalyzed azide–alkyne cycloaddition (CuAAC). Imaging in MCF-7 cells revealed distinct, structure-dependent localization patterns—nuclear for iPUT and iSPD, and extranuclear/mitochondrial for iSPM. Mass spectrometry confirmed that the probes remain chemically intact within cells. Pharmacological inhibition with the polyamine transport blocker AMXT-1501 reduced probe uptake and altered subcellular distribution, confirming engagement with endogenous transport pathways. Pulse–chase experiments further demonstrated dynamic efflux and intercellular transfer of polyamines. In zebrafish larvae, iPAs displayed structure- and tissue-dependent accumulation, with striking differences between neuronal and epidermal cells and among brain regions. Together, these findings establish iPAs as versatile, minimally invasive tools for spatially resolved mapping of polyamine transport and localization in cells and whole organisms. The probes provide a foundation for future studies of polyamine-dependent signaling and metabolism in health and disease.