The docosahexaenoic acid derivatives, diHEP-DPA and TH-DPA, synthesized via recombinant lipoxygenase, ameliorate disturbances in lipid metabolism and liver inflammation in high fat diet-fed mice.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA) and 7S,15R,16S,17S-tetrahydroxy-docosapentaenoic acid (TH-DPA) are two novel lipid mediators derived from docosahexaenoic acid (DHA) that we previously synthesized via combined enzymatic and chemical reactions. In the present study, we investigated the effects of these compounds on disturbances in lipid metabolism and liver inflammation induced by a high fat diet (HFD) in mice. Male BALB/c mice were randomly divided into four groups (n = 10/group): controls, HFD only, HFD + diHEP-DPA, and HFD + TH-DPA. Mice in HFD + diHEP-DPA and HFD + TH-DPA groups were orally administered 20 µg/kg of diHEP-DPA or TH-DPA, respectively. Measurements of adipose accumulation and liver inflammation showed that both diHEP-DPA and TH-DPA decreased adipose tissue mass and liver color depth, as well as total cholesterol, triglycerides, and low-density lipoprotein-cholesterol in the serum of HFD-fed mice compared with mice in the HFD-only group, while elevating high-density lipoprotein-cholesterol. Both of them also decreased hepatic expression of genes encoding lipid synthesis-related proteins (PPARγ, SIRT1, SREBP-1c and FASN) and increased the expression of genes encoding proteins involved in lipid degradation (PPARα and CPT-1) in the liver. Western blotting and quantitative RT-PCR confirmed that diHEP-DPA or TH-DPA administration modulated the expression of inflammation-related genes (TNF-α and IL-6) and inhibited activation of the NF-κB signaling pathway in livers of HFD-fed mice. Taken together, our data indicate that diHEP-DPA and TH-DPA ameliorate liver inflammation and inhibit HFD-induced obesity in mice.

Synthesis of two new lipid mediators from docosahexaenoic acid by combinatorial catalysis involving enzymatic and chemical reaction.

Omega-3 polyunsaturated fatty acids (PUFAs) have been known to have beneficial effects in the prevention of various diseases. Recently, it was identified that the bioactivities of omega-3 are related to lipid mediators, called pro-resolving lipid mediators (SPMs), converted from PUFAs, so they have attracted much attention as potential pharmaceutical targets. Here, we aimed to build an efficient production system composed of enzymatic and chemical catalysis that converts docosahexaenoic acid (DHA) into lipid mediators. The cyanobacterial lipoxygenase, named Osc-LOX, was identified and characterized, and the binding poses of enzyme and substrates were predicted by ligand docking simulation. DHA was converted into three lipid mediators, a 17S-hydroxy-DHA, a 7S,17S-dihydroxy-DHA (RvD5), and a 7S,15R-dihydroxy-16S,17S-epoxy-DPA (new type), by an enzymatic reaction and deoxygenation. Also, two lipid mediators, 7S,15R,16S,17S-tetrahydroxy-DPA (new type) and 7S,16R,17S-trihydroxy-DHA (RvD2), were generated from 7S,15R-dihydroxy-16S,17S-epoxy-DPA by a chemical reaction. Our study suggests that discovering new enzymes that have not been functionally characterized would be a powerful strategy for producing various lipid mediators. Also, this combination catalysis approach including biological and chemical reactions could be an effective production system for the manufacturing lipid mediators.

Synthesis of 13R,20-dihydroxy-docosahexaenoic acid by site-directed mutagenesis of lipoxygenase derived from Oscillatoria nigro-viridis PCC 7112.

Lipoxygenases (LOXs) are implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators involved in immune cell signaling, most of which catalyze peroxidation of polyunsaturated fatty acids by distinct regio- and stereoselectivity. Current reports suggested that conserved amino acid, Gly in R-LOXs and Ala in S-LOXs, in the catalytic domain play an important role in determining the position as well as the stereochemistry of the functional group. Recently, we have confirmed that the catalytic specificity of cyanobacterial lipoxygenase, named Osc-LOX, with alanine at 296 was 13S-type toward linoleic acid, and producing a 17S- hydroxy-docosahexaenoic acid from docosahexaenoic acid (DHA). Here, we aimed to change the catalytic property of LOX from13S-LOX to 9R-LOX by replacing Ala with Gly and to produce a lipid mediators different from the wild-type using DHA. Finally, we succeeded in generating human endogenous a 13R-hydroxy-docosahexaenoic acid and a 13R,20-dihydroxy-docosahexaenoic acid from DHA through an enzymatic reaction using the Osc-LOX-A296G. Our study could enable physiological studies and pharmaceutical research for the 13R,20-dihydroxy-docosahexaenoic acid.

Asymmetric Synthesis of (-)-6-Desmethyl-Fluvirucinine A1 via Conformationally-Controlled Diastereoselective Lactam-Ring Expansions.

The versatile synthesis of (-)-6-desmethyl-fluvirucinine A1 was accomplished at a 24% overall yield through a thirteen-step process from a known vinylpiperidine. The key part involved the elaboration of the distal stereocenters and a macrolactam skeleton via conformationally-induced diastereocontrol and the iterative aza-Claisen rearrangements of lactam precursors.

Spectroscopic methods to analyze drug metabolites.

Drug metabolites have been monitored with various types of newly developed techniques and/or combination of common analytical methods, which could provide a great deal of information on metabolite profiling. Because it is not easy to analyze whole drug metabolites qualitatively and quantitatively, a single solution of analytical techniques is combined in a multilateral manner to cover the widest range of drug metabolites. Mass-based spectroscopic analysis of drug metabolites has been expanded with the help of other parameter-based methods. The current development of metabolism studies through contemporary pharmaceutical research are reviewed with an overview on conventionally used spectroscopic methods. Several technical approaches for conducting drug metabolic profiling through spectroscopic methods are discussed in depth.