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.

Figure 1

Phospholipase-mediated cleavage sites.

Figure 2

Design of NIRF-bearing PLC- and PLA2-cleavable self quenched phospholipids.

Figure 3

Sensitivity of self-quenched fluorescent probes to different phospholipase isoforms as measured by fluorescence release on a plate reader (See Supporting Information).

Scheme 1

Synthesis of Pyro-PtdEtn-BHQ (4), PyroC12-PtdEtn-BHQ (7) and PyroC6-PyroC6-PtdCho (10) and structures for comparison of commercially available self-quenched PLA2-sensitive green fluorescent phospholipid probes PED-6 (11) and B7701 (12). * Attachment sites for fluorophores/quencher. Reagents, conditions (all reactions were performed under Ar in dark) and isolated yields (relative to starting NIRF 1, one arrow indicates one-pot reaction): (a) EDC, DMAP, DCM, rt, 72 h; (b) TFA, DCM, 0 °C, 4 h, 20%; (c) BHQ-3+SU PF6-, TEA, DCM, rt, 12 h, 15%; (d) NHS, EDC, DMAP, DCM, rt, 3 h; (e) H2N(CH2)11CO2H, TEA, DCM, rt, 72 h, 85% (f) 2, EDC, DMAP, DCM, rt, 72 h; (g) TFA, DCM, 0 °C, 4 h, 15%; (h) BHQ-3+SU PF6-, TEA, DCM, rt 12 h, 10%; (i) NHS, EDC, DMAP, DCM, rt, 3 h; (j) H2N(CH2)5CO2H, TEA, DCM, rt, 72 h, 75%; (k) EDC, DMAP, DCM, 40 °C, 85 h, 8%