Synthesis of 13(R)-Hydroxy-7Z,10Z,13R,14E,16Z,19Z Docosapentaenoic Acid (13R-HDPA) and Its Biosynthetic Conversion to the 13-Series Resolvins

Abstract

Specialized pro-resolving lipid mediators are biosynthesized during the resolution phase of acute inflammation from n-3 polyunsaturated fatty acids. Recently, the isolation and identification of the four novel mediators denoted 13-series resolvins, namely, RvT1 (1), RvT2 (2), RvT3 (3) and RvT4 (4), were reported, which showed potent bioactions characteristic for specialized pro-resolving lipid mediators. Herein, based on results from LC/MS-MS metabololipidomics and the stereoselective synthesis of 13(R)-hydroxy-7Z,10Z,13R,14E,16Z,19Z docosapentaenoic acid (13R-HDPA, 5), we provide direct evidence that the four novel mediators 1-4 are all biosynthesized from the pivotal intermediate 5. The UV and LC/MS-MS results from synthetic 13R-HDPA (5) matched those from endogenously and biosynthetically produced material obtained from in vivo infectious exudates, endothelial cells, and human recombinant COX-2 enzyme. Stereochemically pure 5 was obtained with the use of a chiral pool starting material that installed the configuration at the C-13 atom as R. Two stereoselective Z-Wittig reactions and two Z-selective reductions of internal alkynes afforded the geometrically pure alkene moieties in 5. Incubation of 5 with isolated human neutrophils gave all four RvTs. The results presented herein provide new knowledge on the biosynthetic pathways and the enzymatic origin of RvTs 1-4.

Scheme 1. Chemical Structure of 13R-HDPA (5) and Outline of Its Proposed Biosynthesis from n-3 DPA

Scheme 2. Synthesis of (Z)-Hept-4-en-1-yne (10)

Scheme 3. Synthesis of Vinyl Iodide 13

Scheme 4. Synthesis of Wittig Salt 19

Scheme 5. Synthesis of 13R-HDPA (5)

Figure 1

13-HDPA MRM chromatograms from (A) endothelial cells, (B) infectious exudates, (C) hrCOX-2, and (D) synthetic material of 5. (E) Coinjection of endothelial and synthetic 5.

Figure 2

13-HDPA chiral LC-MS-MS derived from (A) endothelial cells, (B) infectious exudates, (C) hrCOX-2, and (D) synthetic material. (E) Coinjection of endothelial and synthetic 13R-HDPA.

Figure 3

MS-MS spectra employed in the identification of 13-HDPA from (A) endothelial cells, (B) infectious exudates, and (C) synthetic material. n = 3 endothelial cell donors, n = 3 mouse exudates, and n = 3 for synthetic material.

Figure 4

UV spectra for (A) hrCOX-2 13-HDPA and (B) synthetic 13R-HDPA (5).

Figure 5

Human neutrophils convert 13R-HDPA (5) to RvT1–4 (1–4). Human neutrophils were isolated from peripheral blood of healthy donors and incubated (2 × 10⁷ cells/mL) with or without 13R-HDPA (5) (45 min, 37 °C, 2 μM A23187, PBS, pH = 7.45). Incubations were quenched with two volumes of ice cold MeOH and products extracted and profiled using lipid mediator metabololipidomics. (A) MRM chromatograms for each of the RvT1–4 with relative abundances to their levels in each of the incubations. (B) MS-MS spectra employed in the identification of RvT1 (1), RvT2 (2), RvT3 (3) and RvT4 (4). Results are representative of n = 3 healthy volunteers.