Enantio- and stereospecific syntheses of 15(R)-Me-PGD2, a potent and selective DP2-receptor agonist.

The first total synthesis of 15(R)-Me-PGD2 3 is reported. The synthesis is based on the enantioselective and stereospecific syntheses of synthon 17 and its attachment to the five-membered ring by a olefin cross metathesis reaction. This approach permits the introduction of a side chain with a predetermined stereogenic center into the prostanoid ring, resulting in the synthesis of 15R-methyl prostaglandin D2 and allows rapid access to other prostanoids.

Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant.

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.

Reductive Deprotection of Silyl Groups with Wilkinson's Catalyst/Catechol Borane.

Traditionally silyl groups are deprotected with acids and fluorides. These methods are, however, less discriminating when multi-silyl groups are present in the same molecule resulting in lower yields of desired products. The manipulation of these functions during the total synthesis of natural products, e.g. prostaglandins and isoprostanes, requires the selective protection and deprotection of these groups.We are reporting here on a mild, selective and efficient method for the reductive deprotection of silyl groups using Wilkinson's catalyst/catechol borane or catechol borane alone.

Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant that is synthesized from the 5-lipoxygenase product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by the NADP+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH), previously reported only in inflammatory cells. Because of their critical location at the interface of the lung with the external environment, we sought to determine whether epithelial cells could also synthesize this substance. We found that HEp-2, T84, A549, and BEAS-2B cells all synthesize 5-oxo-ETE from 5-HETE in amounts comparable to leukocytes. The epithelial dehydrogenase is localized in the microsomal fraction, requires NADP+, and is selective for the S-isomer of 5-HETE, suggesting that it is identical to leukocyte 5-HEDH. Normal human bronchial epithelial cells have an even greater capacity to synthesize 5-oxo-ETE. H2O2 dramatically stimulates its synthesis in association with increased levels of intracellular GSSG and NADP+. These responses were all blocked by removal of GSH/GSSG with N-ethylmaleimide, suggesting that H2O2 stimulates 5-oxo-ETE synthesis by raising NADP+ levels through activation of the GSH redox cycle. Airway smooth muscle cells can also synthesize 5-oxo-ETE, but to a lesser extent. These results suggest that epithelial cells may be a major source of 5-oxo-ETE under conditions of oxidative stress, which may contribute to eosinophil infiltration in allergic diseases.

Agonist and antagonist effects of 15R-prostaglandin (PG) D2 and 11-methylene-PGD2 on human eosinophils and basophils.

Prostaglandin (PG) D2 acts through both the DP(1) receptor, which is coupled to adenylyl cyclase, and the DP2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells), which is present on eosinophils, basophils, and Th2 cells and results in cell activation and migration. The most potent prostanoid DP2 agonist so far reported is 15R-methyl-PGD2, in which the hydroxyl group has the unnatural R configuration. In contrast, the corresponding analog possessing the natural 15S configuration is approximately 75 times less potent. This raised the question of whether the isoprostane 15R-PGD2 might have potent DP2 receptor-mediated biological activity. We therefore chemically synthesized 15R-PGD2 and investigated its biological activity. This compound elicited DP2 receptor-mediated CD11b expression in human basophils and eosinophils and induced actin polymerization and migration in eosinophils with a potency about the same as that of PGD2. In contrast, it had only a weak effect on DP1 receptor-mediated adenylyl cyclase activity in human platelets. We also investigated the effects of modification of the 9-hydroxyl and 11-oxo groups of PGD2. Both PGK2, in which the 9-hydroxyl group is replaced by an oxo group, and 11-deoxy-11-methylene PGD2, in which the 11-oxo group is replaced by a CH2 group, have little or no DP1 or DP2 agonist activity. However, the 11-methylene analog is a DP2 antagonist (IC50, approximately 2 microM). We conclude that 15R-PGD2, which may be generated by oxidative stress, is a potent and selective DP2 agonist and that modification of the 11-oxo group of PGD2 can result in DP2 antagonist activity.

Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells.

The 5-lipoxygenase product 5-oxo-ETE (5-oxo-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that is synthesized from 5-HETE (5-hydroxyeicosatetraenoic acid) by 5-HEDH (5-hydroxyeicosanoid dehydrogenase). In the present study, we found that 5-HEDH activity is induced in U937 monocytic cells by differentiation towards macrophages with PMA and in HL-60 myeloblastic cells by 1,25-dihydroxy-vitamin D3. We used PMA-differentiated U937 cells to investigate further the regulation of 5-HEDH. This enzyme exhibits approx. 10000-fold selectivity for NADP+ over NAD+ as a cofactor for the oxidation of 5-HETE, which is maximal at pH 10.2. In contrast, the reverse reaction (5-oxo-ETE-->5-HETE) is NADPH-dependent and is maximal at pH 6. Although the K(m) for the forward reaction (670 nM) is about twice that for the reverse reaction at neutral pH, the V(max) is approx 8-fold higher. The oxidation of 5-HETE to 5-oxo-ETE is supported by very low concentrations of NADP(+) (K(m) 139 nM), inhibited by NADPH (K(i) 224 nM) and is consistent with a ping-pong mechanism. The amount of 5-oxo-ETE synthesized by 5-HEDH depends on the ratio of NADP+ to NADPH. Exposure of U937 cells to oxidative stress (t-butyl hydroperoxide) increased the ratio of NADP+ to NADPH from approx. 0.08 in resting cells to approx. 3, and this was accompanied by a dramatic increase in 5-HETE oxidation to 5-oxo-ETE. We conclude that differentiation of monocytic cells towards macrophages results in enhanced 5-oxo-ETE synthesis and that the ability of cells to synthesize 5-oxo-ETE is tightly regulated by the ratio of intracellular NADP+ to NADPH.

Metabolism of 5-hydroxy-6,8,11,14-eicosatetraenoic acid by human endothelial cells.

There is increasing evidence that proinflammatory products of the 5-lipoxygenase pathway play an important role in cardiovascular disease. In the present study, we found that human endothelial cells rapidly oxidize the 5-lipoxygenase product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for myeloid cells. 5-Oxo-ETE synthesis is strongly stimulated by oxidative stress. This effect is enhanced following inhibition of the pentose phosphate pathway with dehydroepiandrosterone and is mimicked by diamide, which oxidizes intracellular GSH to GSSG. Conversely, it is blocked by depletion of intracellular GSH/GSSG. The kinetics of H2O2-induced 5-oxo-ETE synthesis by endothelial cells correlate well with changes in the intracellular levels of GSSG and NADP+. These results suggest that exposure of the endothelium to oxidative stress and inflammation could result in the synthesis of 5-oxo-ETE, which could then induce the infiltration of inflammatory cells into the tissue.

Total synthesis of 8,12-iso-iPF3alpha-VI, an EPA-derived isoprostane: stereoselective introduction of the fifth asymmetric center.

[reaction: see text] A new and stereoselective approach for the synthesis of all-syn isoprostanes is reported. This method, which is based on acid-catalyzed Diels-Alder reaction, allows the introduction of the side chain with a predetermined stereochemistry of the hydroxy group. The first total synthesis of an eicosapentaenoic acid (EPA)-derived iP, 8,12-iso-iPF3alpha-VI 10, was performed using this approach.

Synthesis of 15R-PGD2: a potential DP2 receptor agonist.

The first total synthesis of 15R-PGD(2)3 was accomplished. The approach used in this report is also an efficient method to produce 15R-PGE(2). 15R-PGD(2), a potential DP(2) receptor agonist, could be an important novel tool for defining the role of this receptor in inflammatory diseases.