Design and Synthesis of Phospholipase C and A2-Activatable Near-Infrared Fluorescent Smart Probes

• Published in: Bioconjugate chemistry Vol. 21; no. 10; pp. 1724 - 1727
• Main Authors: Popov, Anatoliy V, Mawn, Theresa M, Kim, Soungkyoo, Zheng, Gang, Delikatny, E. James
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.10.2010
• Subjects: Drug Design; Enzyme Assays - methods; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Infrared Rays; Phospholipases A2 - chemistry; Phospholipases A2 - metabolism; Type C Phospholipases - chemistry; Type C Phospholipases - metabolism
• DOI: 10.1021/bc100271v
• ISSN: 1043-1802

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 headgroup 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 was 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.