title: Design and synthesis of phospholipase C and A2-activatable near-infrared fluorescent smart probes.
abstract: 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 A(1), A(2) (PLA(2)), 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 PLA(2) 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 (C(6), C(12)) 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 PyroC(6)-PyroC(6)-PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA(2), Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA(2) was negligible due to steric hindrance at the sn-2 position. In contrast, the C(12)-spacered PyroC(12)-PtdEtn-BHQ demonstrated a remarkable selectivity for PLA(2) and the best relative PLA(2)/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.
authors.affiliation: University of Pennsylvania, Department of Radiology, Philadelphia Pennsylvania 19104, USA. avpopov@mail.med.upenn.edu
authors.forename: Anatoliy V
authors.initials: AV
authors.lastname: Popov
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Mawn
authors.forename: Soungkyoo
authors.initials: S
authors.lastname: Kim
authors.forename: Gang
authors.initials: G
authors.lastname: Zheng
authors.forename: E James
authors.initials: EJ
authors.lastname: Delikatny
mesh_terms: D015195:Drug Design; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry*; D007259:Infrared Rays; D054467:Phospholipases A2 / Q000737:chemistry / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D054467:Phospholipases A2; D010738:Type C Phospholipases