Biological applications of phosphorescent probes for sensing molecular oxygen (O2) and bioimaging have gained popularity, but their choice is rather limited. We describe a family of new heterosubstituted phosphorescent bioprobes based on the Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye. The probes are produced by simple click modification of its para-fluorine atoms with thiols, such as 1/2-thio-glucose, thio-poly(ethylene glycol) (PEG), or cysteamine. The probes were designed to have one cell-targeting moiety and three polar moieties forming a hydrophilic shell. Their chemical synthesis and purification were optimized to produce high reaction yields and easy scale-up. The ability to perform as cell-permeable or -impermeable probes was tuned by the polarity and molecular charge of the bioconjugate. The new PtPFPP derivatives were characterized for their spectral properties and cell-penetrating ability in the experiments with mammalian cell cultures, using a time-resolved fluorescence reader and PLIM imaging detection. Structure–activity relationships were established. Thus, the tri- and tetra-PEGylated structures showed low cell internalization allowing their use as extracellular probes, while cysteamine derivatives performed as efficient intracellular probes. No significant cytotoxicity was observed for all of the probes under the experimental conditions used.

Intracellular Optical Oxygen sensing is a convenient approach for monitoring and imaging molecular oxygen (O2) in biological samples, however existing intracellular O2-sensing probes still have some limitations. This study describes one new phosphorescent hetero-substituted derivative of Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) obtained via two-step thiol click-chemistry. Particularly, thio-glucose (Glc) and thio-methyl-polyethylene-glycol (mPEG) moieties were covalently attached to the phosphorescent dye, producing the trans-di-glucosylated-di-PEGylated derivative PtGlc2PEG2 (trans). In a previous publication, we demonstrated the ability of these short PEG oligomers to drastically reduce the ability of the tetra- or tri-PEGylated conjugates to translocate across the cellular membrane. However, in this study, we show the capability of the trans-di-PEGylated-di-glucosylated conformation to allow intracellular staining and O2 sensing (IcO2) in murine embryonic fibroblasts (MEFs) mammalian cells.