The primary focus of this work was to develop activatable probes suitable for in vivo detection of phospholipase activity. Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A1, A2 (PLA2), C (PLC) and D (PLD) based on their site of action. Here we report the design, synthesis and characterization of self-quenching reporter probes that release fluorescent moieties upon cleavage with PLA2 or PLC. A series of phospholipids were synthesized bearing the NIR fluorophore Pyropheophorbide a (Pyro) at the sn-2 position. Fluorescence quenching was achieved by attachment of either a positively charged Black Hole Quencher-3 (BHQ-3) to the phospholipid head group or another neutral Pyro moiety at the sn-1 position. The specificity to different phospholipases was modulated by insertion of spacers (C6, C12) between Pyro and the lipid backbone. The specificity of the quenched fluorescent phospholipids were assayed on a plate reader against a number of phospholipases and compared with two commercial probes bearing the visible fluorophore BODIPY. While PyroC6-PyroC6-PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA2, Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA2 was negligible due to steric hindrance at the sn-2 position. In contrast, the C12-spacered PyroC12-PtdEtn-BHQ demonstrated a remarkable selectivity for PLA2 and the best relative PLA2/PLC sensitivity, significantly outperforming previously known probes. These results open an avenue for future in vivo experiments and for new probes to detect PL activity.
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