Synthesis, Biological Investigation, and Structural Revision of Sielboldianin A.

The two ar-bisabol sesquiterpenoids (+)-sielboldianin A (1) and (+)-sielboldianin B (2) were isolated from the stem bark of the plant Fraxinus sielboldiana and belong to a medicinally interesting class of natural products used in traditional Chinese medicine. Herein the total synthesis of the proposed structure of (+)-sielboldianin A (1) is reported using an organocatalyzed enantioselective bromolactonization protocol. X-ray analysis of a key intermediate together with specific rotation values and NOESY data of the synthesized product enabled the revision of the absolute configuration of the natural product (+)-sielboldianin A to (7 R,10 R). Studies on the antioxidant effects using two cell-based assays were conducted. These studies revealed that the enantiomer of 1 exhibited antioxidant effects with IC50 values of 18 ± 3 µM in a cellular lipid peroxidation antioxidant activity assay. Moreover, (-)-1 showed strong protective effects against reactive oxygen species in a cell-based antioxidant activity assay (IC50 = 31 ± 5 µM). In addition, the two ar-sesquiterpenoids (-)-boivinianin B and (-)-gossoronol showed no effect in either assay. No cytotoxic activity in the K562 cancer cell line was observed for the three sesquiterpenoids tested (IC50 > 50 µM).

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

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.

The Protectin PCTR1 Is Produced by Human M2 Macrophages and Enhances Resolution of Infectious Inflammation.

Inflammation and its natural resolution are host-protective responses triggered by infection or injury. The resolution phase of inflammation is regulated by enzymatically produced specialized pro-resolving mediators. We recently identified a new class of peptide-conjugated specialized pro-resolving mediators that carry potent tissue regenerative actions that belong to the protectin family and are coined protectin conjugates in tissue regeneration (PCTR). Herein, with the use of microbial-induced peritonitis in mice and liquid chromatography-tandem mass spectrometry-based lipid mediator metabololipidomics, we found that PCTR1 is temporally regulated during self-resolving infection. When administered at peak of inflammation, PCTR1 enhanced macrophage recruitment and phagocytosis of Escherichia coli, decreased polymorphonuclear leukocyte infiltration, and counter-regulated inflammation-initiating lipid mediators, including prostaglandins. In addition, biologically produced PCTR1 promoted human monocyte and macrophage migration in a dose-dependent manner (0.001 to 10.0 nmol/L). We prepared PCTR1 via organic synthesis and confirmed that synthetic PCTR1 increased macrophage and monocyte migration, enhanced macrophage efferocytosis, and accelerated tissue regeneration in planaria. With human macrophage subsets, PCTR1 levels were significantly higher in M2 macrophages than in M1 phenotype, along with members of the resolvin conjugates in tissue regeneration and maresin conjugate families. In contrast, M1 macrophages gave higher levels of cysteinyl leukotrienes. Together, these results demonstrate that PCTR1 is a potent monocyte/macrophage agonist, regulating key anti-inflammatory and pro-resolving processes during bacterial infection.

Synthesis of the 16S,17S-Epoxyprotectin Intermediate in the Biosynthesis of Protectins by Human Macrophages.

The n-3 polyunsaturated fatty acids act as substrates during the resolution phase of acute inflammation for the biosynthesis of specialized pro-resolving lipid mediators. One premier example is the C22-dihydroxy-polyunsaturated fatty acid protectin D1 (1). The human 15-lipoxygenase type I, via stereoselective processes and with docosahexaenoic acid as the substrate, enables the formation of this specialized pro-resolving lipid mediator. Herein, based on results from LC/MS-MS metabololipidomics, support is presented for the apprehended biosynthesis of 1 in human macrophages occurring via the intermediate 16S,17S-epoxyprotectin (5). Stereochemically pure 5 was obtained using the Katsuki-Sharpless epoxidation protocol, establishing the chirality at the C16 and C17 atoms, one Z-selective reduction, and E- and Z-stereoselective Wittig reactions. In addition, information on the nonenzymatic aqueous hydrolysis products and the half-life of 16S,17S-epoxyprotectin (5) is presented.

Synthesis and anti-inflammatory and pro-resolving activities of 22-OH-PD1, a monohydroxylated metabolite of protectin D1.

Protectin D1 (PD1 (3)), a C22-dihydroxylated polyunsaturated fatty acid biosynthesized from all-Z-docosahexaenoic acid, belongs to the new family of endogenous mediators referred to as specialized pro-resolving lipid mediators. PD1 (3) is a natural product that displays potent anti-inflammatory properties together with pro-resolving actions including inhibition of polymorphonuclear leukocyte (PMN) infiltration and promotion of macrophage phagocytosis and efferocytosis. Given its potent endogenous actions, this compound has entered several clinical development programs. Little has been reported on the metabolism of PD1 (3). The synthesis and biological evaluations of the ω-22 monohydroxylated metabolite of PD1 (3), named herein 22-OH-PD1 (6), are presented. LC-MS/MS data of the free acid 6, obtained from hydrolysis of the synthetic methyl ester 7, matched data for the endogenously produced 22-OH-PD1 (6). Compound 6 exhibited potent pro-resolving actions by inhibiting PMN chemotaxis in vivo and in vitro comparable to its precursor PD1 (3) and decreased pro-inflammatory mediator levels in inflammatory exudates. The results reported herein provide new knowledge of the metabolism of the protectin class of specialized pro-resolving mediators.

Total synthesis of the anti-inflammatory and pro-resolving lipid mediator MaR1n-3 DPA utilizing an sp(3) -sp(3) Negishi cross-coupling reaction.

The first total synthesis of the lipid mediator MaR1n-3 DPA (5) has been achieved in 12 % overall yield over 11 steps. The stereoselective preparation of 5 was based on a Pd-catalyzed sp(3) -sp(3) Negishi cross-coupling reaction and a stereocontrolled Evans-Nagao acetate aldol reaction. LC-MS/MS results with synthetic material matched the biologically produced 5. This novel lipid mediator displayed potent pro-resolving properties stimulating macrophage efferocytosis of apoptotic neutrophils.

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

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.