Biological Evaluations, NMR Analyses, Molecular Modeling Studies, and Overview of the Synthesis of the Marine Natural Product (-)-Mucosin.

Natural products obtained from marine organisms continue to be a rich source of novel structural architecture and of importance in drug discovery, medicine, and health. However, the success of such endeavors depends on the exact structural elucidation and access to sufficient material, often by stereoselective total synthesis, of the isolated natural product of interest. (-)-Mucosin (1), a fatty acid derivative, previously presumed to contain a rare cis-bicyclo[4.3.0]non-3-ene moiety, has since been shown to be the trans-congener. Analytically, the fused bicyclic ring system in (-)-1 constitutes a particular challenge in order to establish its relative and absolute stereochemistry. Herein, data from biological evaluations, NMR and molecular modeling studies of (-)-1 are presented. An overview of the synthetic strategies enabling the exact structural elucidation of (-)-mucosin (1) is also presented.

Stereoselective Synthesis, Pro-resolution, and Anti-inflammatory Actions of RvD5n-3 DPA.

The methyl ester of resolvin D5n-3 DPA, a lipid mediator biosynthesized from the omega-3 fatty acid n-3 docosapentaenoic acid, was stereoselectively prepared in 8% yield over 12 steps (longest linear sequence). The key steps for the introduction of the two stereogenic secondary alcohols were an organocatalyzed oxyamination and the Midland Alpine borane reduction. For the assembly of the carbon chain, the Sonogashira cross-coupling reaction and the Takai olefination were utilized. The physical properties, including retention time in liquid chromatography and tandem mass spectra, of the synthetic material were matched against material from human peripheral blood and mouse infectious exudates. Synthetic RvD5n-3 DPA, obtained just prior to biological experiments, displayed potent leukocyte-directed activities, upregulating the ability of neutrophils and macrophages to phagocytose bacteria, known as hallmark bioactions of specialized pro-resolving endogenous mediators.

Expression and function of resolvin RvD1n-3 DPA receptors in oral epithelial cells.

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1n-3 DPA , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1n-3 DPA exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1n-3 DPA on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1n-3 DPA induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1n-3 DPA is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

Stereoselective Synthesis, Configurational Assignment and Biological Evaluations of the Lipid Mediator RvD2n-3 DPA.

Herein we report the first total synthesis of RvD2n-3 DPA , an endogenously formed mediator biosynthesized from the omega-3 fatty acid n-3 docosapentaenoic acid. The key steps are the Midland Alpine borane reduction, Sonogashira cross-coupling reactions, and a Z-selective alkyne reduction protocol, yielding RvD2n-3 DPA methyl ester in 13 % yield over 12 steps (longest linear sequence). The physical property data (UV chromophore, chromatography and MS/MS fragmentation) of the synthetic lipid mediator matched those obtained from biologically produced material. Moreover, synthetic RvD2n-3 DPA also carried the potent biological activities of enhancing macrophage uptake of Staphylococcus aureus and zymosan A bioparticles.

Some Biogenetic Considerations Regarding the Marine Natural Product (-)-Mucosin.

Recently, the identity of the marine hydrindane natural product (-)-mucosin was revised to the trans-fused structure 6, thereby providing a biogenetic puzzle that remains to be solved. We are now disseminating some of our insights with regard to the possible machinery delivering the established architecture. Aspects with regard to various modes of cyclization in terms of concerted versus stepwise processes are held up against the enzymatic apparatus known to be working on arachidonic acid (8). To provide a contrast to the tentative polyunsaturated fatty acid biogenesis, the structural pattern featured in (-)-mucosin (6) is compared to some marine hydrinane natural products of professed polyketide descent. Our appraisal points to a different origin and strengthens the hypothesis of a polyunsaturated fatty acids (PUFA) as the progenitor of (-)-mucosin (6).

Organoselenium Accelerated Bromolactonization Reaction.

A highly efficient and regioselective bromolactonization protocol is reported. The quantitative formation of synthetically versatile bromolactones occurs in the presence of only 0.1 mol % of an organoselenium compound, coined DECAD herein, within 90 min. DECAD is conveniently prepared on multigram scale from cheap racemic camphor. The presented protocol was easy to scale up and performed equally well on gram scale. Investigation of the mechanism revealed that DECAD forms a selenonium ylene in situ.

Early increase of specialized pro-resolving lipid mediators in patients with ST-elevation myocardial infarction.

BACKGROUND: Termination of acute inflammation is an active process orchestrated by lipid mediators (LM) derived from polyunsaturated fatty acids, referred to as specialized pro-resolving mediators (SPM). These mediators also provide novel therapeutic opportunities for treating inflammatory disease. However, the regulation of these molecules following acute myocardial infarction (MI) remains of interest. METHODS: In this prospective observational study we aimed to profile plasma levels of SPMs in ST-elevation MI (STEMI) patients during the first week following MI. Plasma LM concentrations were measured in patients with STEMI (n = 15) at three time points and compared with stable coronary artery disease (CAD; n = 10) and healthy controls (n = 10). FINDINGS: Our main findings were: (i) Immediately after onset of MI and before peak troponin T levels, STEMI patients had markedly increased levels of SPMs as compared with healthy controls and stable CAD patients, with levels of these mediators declining during follow-up. (ii) The increase in SPMs primarily reflected an increase in docosapentaenoic acid- and docosahexaenoic acid-derived protectins. (iii) Several individual protectins were correlated with the rapid increase in neutrophil counts, but not with CRP. (iv) A shift in 5-LOX activity from the leukotriene B4 pathway to the pro-resolving RvTs was observed. INTERPRETATION: The temporal regulation of SPMs indicates that resolution mechanisms are activated early during STEMI as part of an endogenous mechanism to initiate repair. Thus strategies to boost the activity and/or efficacy of these endogenous mechanisms may represent novel therapeutic opportunities for treatment of patients with MI. FUND: This work was supported by grants from the South-Eastern Norwegian regional health authority, the European Research Council under the European Union's Horizon 2020 research and innovation program, a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society, and the Barts Charity.

Total Synthesis of (-)-Mucosin and Revision of Structure.

The first total synthesis of (-)-mucosin (6), an unusual marine hydrindane natural product incorporating a prostaglandin-like submotif, has been achieved. As a result of the campaign, three of the four all-carbon stereocenters in the purported structure 1 have been revised. Of particular note is the excellent control over ß-chirality in conjugate addition to ester (-)-22 and the facial selectivity in the subsequent protonation of an intermediate silyl ketene acetal.

n-3 Docosapentaenoic acid-derived protectin D1 promotes resolution of neuroinflammation and arrests epileptogenesis.

Epilepsy therapy is based on drugs that treat the symptoms rather than the underlying mechanisms of the disease (epileptogenesis). There are no treatments for preventing seizures or improving disease prognosis, including neurological comorbidities. The search of pathogenic mechanisms of epileptogenesis highlighted that neuroinflammatory cytokines [i.e. interleukin-1ß (IL-1ß), tumour necrosis factor-α (Tnf-α)] are induced in human and experimental epilepsies, and contribute to seizure generation in animal models. A major role in controlling the inflammatory response is played by specialized pro-resolving lipid mediators acting on specific G-protein coupled receptors. Of note, the role that these pathways have in epileptogenic tissue remains largely unexplored. Using a murine model of epilepsy, we show that specialized pro-resolving mechanisms are activated by status epilepticus before the onset of spontaneous seizures, but with a marked delay as compared to the neuroinflammatory response. This was assessed by measuring the time course of mRNA levels of 5-lipoxygenase (Alox5) and 15-lipoxygenase (Alox15), the key biosynthetic enzymes of pro-resolving lipid mediators, versus Il1b and Tnfa transcripts and proteins. In the same hippocampal tissue, we found a similar delayed expression of two main pro-resolving receptors, the lipoxin A4 receptor/formyl peptide receptor 2 and the chemerin receptor. These receptors were also induced in the human hippocampus after status epilepticus and in patients with temporal lobe epilepsy. This evidence supports the hypothesis that the neuroinflammatory response is sustained by a failure to engage pro-resolving mechanisms during epileptogenesis. Lipidomic LC-MS/MS analysis showed that lipid mediator levels apt to resolve the neuroinflammatory response were also significantly altered in the hippocampus during epileptogenesis with a shift in the biosynthesis of several pro-resolving mediator families including the n-3 docosapentaenoic acid (DPA)-derived protectin D1. Of note, intracerebroventricular injection of this mediator during epileptogenesis in mice dose-dependently reduced the hippocampal expression of both Il1b and Tnfa mRNAs. This effect was associated with marked improvement in mouse weight recovery and rescue of cognitive deficit in the novel object recognition test. Notably, the frequency of spontaneous seizures was drastically reduced by 2-fold on average and the average seizure duration was shortened by 40% after treatment discontinuation. As a result, the total time spent in seizures was reduced by 3-fold in mice treated with n-3 DPA-derived protectin D1. Taken together, the present findings demonstrate that epilepsy is characterized by an inadequate engagement of resolution pathways. Boosting endogenous resolution responses significantly improved disease outcomes, providing novel treatment avenues.

PDn-3 DPA Pathway Regulates Human Monocyte Differentiation and Macrophage Function.

Macrophages are central in orchestrating the clearance of apoptotic cells and cellular debris during inflammation, with the mechanism(s) regulating this process remaining of interest. Herein, we found that the n-3 docosapentaenoic acid-derived protectin (PDn-3 DPA) biosynthetic pathway regulated the differentiation of human monocytes, altering macrophage phenotype, efferocytosis, and bacterial phagocytosis. Using lipid mediator profiling, human primary cells and recombinant enzymes we found that human 15-lipoxygenases initiate the PDn-3 DPA pathway catalyzing the formation of an allylic epoxide. The complete stereochemistry of this epoxide was determined using stereocontrolled total organic synthesis as 16S,17S-epoxy-7Z,10Z,12E,14E,19Z-docosapentaenoic acid (16S,17S-ePDn-3 DPA). This intermediate was enzymatically converted by epoxide hydrolases to PD1n-3 DPA and PD2n-3 DPA, with epoxide hydrolase 2 converting 16S,17S-ePDn-3 DPA to PD2n-3 DPA in human monocytes. Taken together these results establish the PDn-3 DPA biosynthetic pathway in human monocytes and macrophages and its role in regulating macrophage resolution responses.

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).

Protectin D1n-3 DPA and resolvin D5n-3 DPA are effectors of intestinal protection.

The resolution of inflammation is an active process orchestrated by specialized proresolving lipid mediators (SPM) that limit the host response within the affected tissue; failure of effective resolution may lead to tissue injury. Because persistence of inflammatory signals is a main feature of chronic inflammatory conditions, including inflammatory bowel diseases (IBDs), herein we investigate expression and functions of SPM in intestinal inflammation. Targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics was used to identify SPMs from n-3 polyunsaturated fatty acids in human IBD colon biopsies, quantifying a significant up-regulation of the resolvin and protectin pathway compared with normal gut tissue. Systemic treatment with protectin (PD)1n-3 DPA or resolvin (Rv)D5n-3 DPA protected against colitis and intestinal ischemia/reperfusion-induced inflammation in mice. Inhibition of 15-lipoxygenase activity reduced PD1n-3 DPA and augmented intestinal inflammation in experimental colitis. Intravital microscopy of mouse mesenteric venules demonstrated that PD1n-3 DPA and RvD5n-3 DPA decreased the extent of leukocyte adhesion and emigration following ischemia-reperfusion. These data were translated by assessing human neutrophil-endothelial interactions under flow: PD1n-3 DPA and RvD5n-3 DPA reduced cell adhesion onto TNF-α-activated human endothelial monolayers. In conclusion, we propose that innovative therapies based on n-3 DPA-derived mediators could be developed to enable antiinflammatory and tissue protective effects in inflammatory pathologies of the gut.

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.

Selective identification of specialized pro-resolving lipid mediators from their biosynthetic double di-oxygenation isomers.

The n-3 polyunsaturated fatty acids are substrates for lipoxygenases and cyclooxygenases. During inflammatory processes, these enzymes form several distinct families of oxygenated polyunsaturated fatty acids coined specialized pro-resolving lipid mediators. Structural elucidation of these natural products using LC-MS/MS based metabololipidomics with the pico- to nanogram amounts of biosynthetic material available have been performed. The specialized pro-resolving lipid mediators display stereospecific and potent anti-inflammatory and pro-resolving actions. Most often the different families among these mediators are chemically characterized by two or three chiral, secondary alcohols, separated by either anE,E,Z-triene or an E,Z,E,E-tetraenemoiety. The lipoxygenases also form other oxygenated polyunsaturated natural products, coined double di-oxygenation products, that are constitutional isomers of the protectin and maresin families of specialized pro-resolving lipid mediators. Very often these products exhibit similar chromatographic properties and mass spectrometrical fragment ions as the pro-resolving mediators. In addition, the double di-oxygenation products are sometimes formed in larger amounts than the specialized pro-resolving lipid mediators. Thus, it is not always possible to distinguish between the specialized pro-resolving mediators and their double di-oxygenation isomers in biological systems, using LC/MS-based techniques. Herein, a convenient and easy-to-use protocol to meet this challenge is presented.

Total synthesis based on the originally claimed structure of mucosin.

The first total synthesis aimed at the naturally occurring eicosanoid bicycle mucosin is reported. A practical route has been devised allowing the issues relating to the previous assignment of stereochemistry to be examined. X-ray crystallography was performed on a late stage intermediate to pinpoint the topological relationship displayed by the featured bicyclo[4.3.0]non-3-ene scaffold.

Enantioselective Organocatalyzed Bromolactonizations: Applications in Natural Product Synthesis.

Asymmetric bromolactonization reactions of δ-unsaturated carboxylic acids have been investigated in the presence of 10 chiral squaramide hydrogen-bonding organocatalysts. The best catalyst enabled the cyclization of several 5-arylhex-5-enoic acids into the corresponding bromolactones with up to 96% ee and in high to excellent chemical yields. The reported catalysts are prepared in a straightforward manner in two steps from dimethyl squarate. The utility of the developed protocol was demonstrated in highly enantioselective syntheses of the sesquiterpenoids (-)-gossoronol and (-)-boivinianin B. Both natural products were obtained in ≥99% enantiomeric excess.

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.

Dienals derived from pyridinium salts and their subsequent application in natural product synthesis.

Transformation of quaternary pyridinium compounds into functionalized conjugated dienes can be adapted to natural product synthesis with great effect. Most conspicuously, the transformation has been employed in the preparation of polyenic structures. However, in a more convoluted application, polycyclic systems have arisen from elaboration of the diene motif. The goal of the present account is to survey the utility of dienals derived from pyridinium salts as the means to establish molecular architecture featured in natural products.