Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

Cationic guanidinium and phosphonium functionalized cytosine morpholino tetramer (G3) and trimer (P3) have been reported for the first time. They show efficient cellular uptake properties in full growth medium, quantified by fluorescence activated cell sorting (FACS) and visualized by fluorescence microscopy. An interesting feature of these transporters is their localization in the nucleolus or the rest of nucleus, determined by the nature of cationic moieties attached with cytosine base. Co‐staining with MitoTracker Deep Red depicts their ability to penetrate mitochondrial membrane. Fluorescence imaging displays their uniform distribution throughout zebrafish larvae with no significant toxicity. FACS analysis at 4°C indicated that transfection of G3 transporter is energy dependent whereas P3 is energy independent. When covalently conjugated, G3 is capable of transporting a 25‐mer phosphorodiamidate morpholino oligomer (PMO) into the cells. It exhibits antisense effect against Gli1 gene in hedgehog pathway, confirmed in preliminary β‐glactosidase assay performed in Ptch1–/– cells. Further antisense effect was confirmed when G3‐conjugated zebrafish Gli1‐PMO was injected into embryo to obtain Gli1‐dependent phenotypes. To the best of our knowledge, herein, we first report that molecular transporters with a minimum number of three cationic groups are capable of efficiently passing through plasma, nuclear and mitochondrial membranes and show distribution in zebrafish larvae. Synthesis and cellular internalization studies of cationic guanidinium and phosphonium functionalized cytosine morpholino oligomers, G3 and P3 have been reported, respectively. These transporters have the ability to penetrate nuclear and mitochondrial membranes in complete growth medium. They exhibit uniform distribution in zebrafish larvae. G3 has the ability to transport antisense morpholino oligos in Ptch1‐/‐ cells and zebrafish embryos.