Total synthesis of the lipid mediator PD1n-3 DPA: configurational assignments and anti-inflammatory and pro-resolving actions

Abstract

The polyunsaturated lipid mediator PD1n-3 DPA (5) was recently isolated from self-resolving inflammatory exudates of 5 and human macrophages. Herein, the first total synthesis of PD1n-3 DPA (5) is reported in 10 steps and 9% overall yield. These efforts, together with NMR data of its methyl ester 6, confirmed the structure of 5 to be (7Z,10R,11E,13E,15Z,17S,19Z)-10,17-dihydroxydocosa-7,11,13,15,19-pentaenoic acid. The proposed biosynthetic pathway, with the involvement of an epoxide intermediate, was supported by results from trapping experiments. In addition, LC-MS/MS data of the free acid 5, obtained from hydrolysis of the synthetic methyl ester 6, matched data for the endogenously produced biological material. The natural product PD1n-3 DPA (5) demonstrated potent anti-inflammatory properties together with pro-resolving actions stimulating human macrophage phagocytosis and efferocytosis. These results contribute new knowledge on the n-3 DPA structure-function of the growing numbers of specialized pro-resolving lipid mediators and pathways.

Figure 1

Proposed biosynthesis of PD1_n-3 DPA (5).

Scheme 1. Synthesis of Wittig Salt 7 and Intermediate 14

Scheme 2. Final Steps of the Synthesis of PD1_n-3 DPA (5)

Figure 2

Endogenous PD1_n-3 DPA (5) from human macrophages and resolving inflammatory exudates match synthetic material. MRM chromatograms for selected ion pair m/z 361–183 depicting (A) PD1_n-3 DPA (5) from human macrophages (5 × 10⁷ cells/mL) incubated with 0.1 mg of opsonized zymosan and n-3 DPA (1 μM, 37 °C, 30 min, DPBS^+/+, pH = 7.45). Results are representative of n = 3 human macrophage preparations. (B) Endogenous PD1_n-3 DPA (5) obtained from mice injected with zymosan (1 mg/mouse) and exudates collected at 4 h. Results are representative of n = 4 mice exudates. (C) Synthetic material (inset: characteristic UV-absorption spectrum, λ_max^MeOH ± 1 nm). (D) Co-injection of resolving exudate endogenous PD1_n-3 DPA (5) with synthetic material. Results are representative of n = 4.

Figure 3

PD1_n-3 DPA (5) displays potent anti-inflammatory and pro-resolving actions. PD1_n-3 DPA, (5), PD1 (3), or vehicle (saline containing 0.01% EtOH) were administered iv 5 min prior to ip administration of zymosan (1 mg). Exudates were collected at 4 h, and the number of infiltrated neutrophils was determined by flow cytometry (top right inset) and light microscopy. Results are mean ± SEM. n = 4 mice per treatment (**p < 0.01 vs vehicle group). (B, C) Macrophages were incubated with vehicle (0.1% EtOH in PBS), PD1_n-3 DPA (5) (100 nM to 10 pM), or PD1 (3) (100 nM to 10 pM; 15 min, 37 °C, pH = 7.45) prior to addition of (B) fluorescently labeled zymosan (1:10 macrophages to zymosan) or (C) fluorescently labeled apoptotic human neutrophils. After 60 min (37 °C, pH = 7.45), the incubation was stopped, extracellular fluorescence quenched using trypan blue, and phagocytosis assessed using a SpectraMax M3 plate reader. Results are mean ± SEM. n = 4 macrophage preparations (*p < 0.05 vs PBS-incubated macrophages).