Title: Efficient Synthesis of Stearidonic Acid Enriched Triacylglycerol from Ahiflower Seed Oil via a Two-Step Enzyme Reaction Ju C, Lee YJ, Yoon HS, Kim BH, Kim IH Ref: J Oleo Sci, 71:1679, 2022 : PubMed
Stearidonic acid (SDA) is a plant-based n-3 polyunsaturated fatty acid with multiple biological activities. The enrichment of SDA and synthesis of triacylglycerol (TAG) were carried out consecutively via two lipase-catalyzed reactions, hydrolysis, and esterification. First, SDA was enriched into a glyceride fraction from ahiflower seed oil by Candida rugosa lipase-catalyzed hydrolysis. Under the optimum conditions of 35 degreesC, 0.1% lipase powder of Lipase OF, and 50% buffer solution (based on the weight of total substrate), SDA was enriched from 21.6 to 40.7 wt% in glyceride fraction. SDA-enriched TAG was then synthesized from the SDA-enriched glyceride and the SDA-enriched fatty acid via esterification using an in-house immobilized lipase as a biocatalyst. The SDA-enriched fatty acid was obtained from part of the SDA-enriched glyceride by saponification and the in-house immobilized lipase was prepared from Eversa((a)) Transform 2.0 using Lewatit VP OC 1600 as a carrier. The optimum reaction conditions for the synthesis of TAG were a temperature of 50 degreesC, an enzyme loading of 10%, and a vacuum of 10 mmHg. A maximum conversion to TAG of ca. 94% was achieved after 12 h under the optimum conditions.
Title: Enzymatic structural modification of monogalactosyldiacylglycerols for potential modulation of hydrophile-lipophile balance Roh S, Lee S, Kim IH, Hee Kim B Ref: Food Chem, 385:132705, 2022 : PubMed
This study aimed to enzymatically prepare structured monogalactosyldiacylglycerols (MGDGs) with different hydrophile-lipophile balance (HLB) values for use as emulsifiers. Acidolysis of Perilla frutescens-derived MGDGs with capric acid (10:0) was conducted to obtain structured MGDGs containing 10:0. Lewatit VP OC 1600-immobilized Rhizomucor miehei lipase was used as the biocatalyst. Structured MGDGs (HLB value = 2.95-7.17) containing 13.0-70.6 mol% 10:0 were obtained from P. frutescens MGDGs (HLB value = 1.93). A quadratic regression equation (R(2) = 0.920) to predict the 10:0 content of the structured MGDGs under the given conditions was established using response surface methodology. Using a linear regression equation (R(2) = 0.999) to predict the HLB value by 10:0 content, structured MGDGs containing 27.1-54.6 mol% 10:0 were predicted to have an HLB value of 4-6, indicating their potential applicability as hydrophobic emulsifiers. Structured MGDGs with a purity of - 43% w/w were obtained from the reaction products using silica column chromatography.
Title: Production of stearidonic acid-rich triacylglycerol via a two-step enzymatic esterification Kim NH, Kim H, Choi N, Kim Y, Kim BH, Kim IH Ref: Food Chem, 270:332, 2019 : PubMed
The aim of this study was to synthesize stearidonic acid (SDA)-rich triacylglycerol (TAG) via a two-step lipase-catalyzed esterification under vacuum. SDA-rich fatty acid, which was prepared from echium oil via Candida rugosa lipase-catalyzed selective esterification, was used as the substrate. Two different immobilized lipases, Novozym 435 from Candida antarctica and Lipozyme TL IM from Thermomyces lanuginosus, were employed for the synthesis of SDA-rich TAG. In the first step, Novozym 435-catalyzed esterification of the SDA-rich fatty acid with glycerol was carried out for 2 h. In the second step, Lipozyme TL IM-catalyzed esterification of the reaction mixture from the first step was performed for an additional 10 h. The optimal reaction conditions for the second step were a temperature of 65 degreeC, an enzyme loading of 20%, and a vacuum of 0.7 kPa. Consequently, the maximum TAG conversion of ca. 86.4 wt% was obtained after 12 h via a two-step lipase-catalyzed esterification.
Title: Preparation of High Purity delta5-Olefinic Acids from Pine Nut Oil via Repeated Lipase-Catalyzed Esterification Kim H, Choi N, Kim HR, Lee J, Kim IH Ref: J Oleo Sci, 67:1435, 2018 : PubMed
delta5-Olefinic acids have been characterized in gymnosperm plants and have been reported to have several biological health benefits. delta5-Olefinic acids from pine nut oil were effectively concentrated by repeated lipase-catalyzed esterification. The pine nut oil contained three major delta5-olefinic acids, namely taxoleic acid (C18:2 delta5,9), pinolenic acid (C18:3 delta5,9,12), and sciadonic acid (C20:3 delta5,11,14). The fatty acids present in pine nut oil were selectively esterified with ethanol using Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. The delta5-olefinic acids were concentrated in the unesterified fatty acid fraction. The optimum molar ratio of the substrates (fatty acid:ethanol), temperature, the enzyme loading, and the reaction time were 1:7, 25 degC, 5% of total substrate weight, and 6 h, respectively. There was no significant effect in the concentration of delta5-olefinic acids when water was added in the reaction mixture. The same protocol and optimum conditions were employed for two times repeated lipase-catalyzed esterifications. In first lipase-catalyzed esterification, the delta5-olefinic acids content in the pine nut oil increased from 17 mol% to 51 mol% with a yield of 40 mol%. In a second lipase-catalyzed esterification, with the delta5-olefinic acids-concentrated fatty acids obtained from the first reaction as the substrate, the delta5-olefinic acids content increased to 86 mol% with a yield of 15 mol%. Finally, a maximum delta5-olefinic acids content of ca. 96 mol% with a yield of 6 mol% was obtained via a third lipase-catalyzed esterification.
Title: Synthesis of Fatty Acid Methyl Esters Using Mixed Enzyme in a Packed Bed Reactor Ryu J, Choi N, Kim H, Kim BH, Kim HR, Kim IH Ref: J Oleo Sci, 67:321, 2018 : PubMed
Fatty acid methyl esters were synthesized from palm fatty acid distillate (PFAD) and methanol in a packed bed reactor via lipase-catalyzed esterification. The PFAD consisted of 91 wt% of free fatty acids, 2 wt% monoacylglycerides, 3 wt% diacylglycerides, and 4 wt% triacylglycerides. t-Butanol was employed as a reaction medium and a mixed enzyme consisting of Lipozyme TL IM from Thermomyces lanuginosus and Novozym 435 from Candida antarctica was employed as the biocatalyst. The effect of mixed enzyme was investigated and the optimum blending ratio (w/w) of Novozym 435 to Lipozyme TL IM was 5:95. Using the mixed enzyme, the optimum molar ratio (PFAD to methanol) and temperature were determined to be 1:6 and 30 degC, respectively. Under the optimized conditions, the maximum yield of ca. 96% was achieved.
The ability to rapidly detect, identify, and monitor chemical warfare agents (CWAs) is imperative for both military and civilian defense. Since most CWAs and their simulants have an organophosphonate group, which is a hydrogen (H)-bond acceptor, many H-bond donors have been developed to effectively bind to the organophosphonate group. Although thioureas have been actively studied as an organocatalyst, they are relatively less investigated in CWA detection. In addition, there is a lack of studies on the structure-property relationship for gas phase detection. In this study, we synthesized various thioureas of different chemical structures, and tested them for sensing dimethylmethylphosphonate (DMMP), a CWA simulant. Molecular interaction between DMMP and thiourea was measured by (1)H NMR titration and supported by density functional theory (DFT) calculations. Strong H-bond donor ability of thiourea may cause self-aggregation, and CH-pi interaction can play an important role in the DMMP detection. Gas-phase adsorption of DMMP was also measured using a quartz crystal microbalance (QCM) and analyzed using the simple Langmuir isotherm, showing the importance of structure-induced morphology of thioureas on the surface.
Title: Synthesis of alpha-linolenic acid-rich triacylglycerol using a newly prepared immobilized lipase Kim H, Choi N, Oh SW, Kim Y, Hee Kim B, Kim IH Ref: Food Chem, 237:654, 2017 : PubMed
An alpha-linolenic acid (ALA)-rich triacylglycerol (TAG) was synthesized from an ALA-rich fatty acid (FA) from perilla oil and glycerol, using a newly prepared immobilized lipase under vacuum. The ALA-rich FA (purity >90wt%) used as the substrate was prepared by urea complexation from perilla oil FAs. Liquid Lipozyme TL 100L lipase from Thermomyces lanuginosus was used for immobilization. Nine different hydrophilic and hydrophobic carriers for immobilization were tested, and Duolite A568, which is a hydrophilic resin, was selected as the best carrier. This immobilized lipase was used to synthesize TAG by direct esterification under vacuum. The parameters investigated were temperature, enzyme loading, and vacuum level. The optimum reaction conditions were a temperature of 60 degC, an enzyme loading of 15% (based on the total weight of the substrate), and a vacuum of 0.7kPa, respectively. The maximum conversion to TAG of ca. 88wt% was obtained in 12h under the optimum conditions.
Title: Production of Biodiesel from Acid Oil via a Two-Step Enzymatic Transesterification Choi N, Lee JS, Kwak J, Lee J, Kim IH Ref: J Oleo Sci, 65:913, 2016 : PubMed
A two-step enzymatic transesterification process in a solvent-free system has been developed as a novel approach to the production of biodiesel using acid oil from rice bran oil soapstock. The acid oil consisted of 53.7 wt% fatty acids, 2.4 wt% monoacylglycerols, 9.1 wt% diacylglycerols, 28.8 wt% triacylglycerols, and 6.0 wt% others. Three immobilized lipases were evaluated as potential biocatalysts, including Novozym 435 from Candida antarctica, Lipozyme RM IM from Rhizomucor miehei, and Lipozyme TL IM from Thermomyces lanuginosus. The effects of molar ratio of acid oil to ethanol, temperature, and enzyme loading were investigated to determine the optimum conditions for the transesterification with the three immobilized lipases. The optimum conditions of the three immobilized lipases were a molar ratio of 1:5 (acid oil to ethanol), the temperature range of 30-40 degC, and the enzyme loading range of 5-10%. The two-step transesterification was then conducted under the optimum conditions of each lipase. The stepwise use of Novozym 435 and Lipozyme TL IM or Lipozyme RM IM and Lipozyme TL IM resulted in similar or higher levels of yield to the individual lipases. The maximum yields obtained in both stepwise uses were ca. 92%.
Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.
Title: Preparation of Highly Purified Stearidonic Acid from Echium Oil via an Enzymatic Method Combined with Preparative High Performance Liquid Chromatography Baik JY, Kim NH, Oh SW, Kim IH Ref: J Oleo Sci, 64:729, 2015 : PubMed
Stearidonic acid (SDA), an n-3 polyunsaturated fatty acid (PUFA), can be obtained from plant origin oils and it can be a good source of PUFA for vegetarians. SDA can be easily converted to longer PUFA such as docosahexaenoic acid and eicosapentaenoic acid. Highly purified stearidonic acid (SDA) was prepared successfully from echium oil via an enzymatic method combined with preparative high performance liquid chromatography. In the 1(st) step, SDA enrichment was accomplished using Candida rugosa lipase and 39.5% of SDA was obtained in the fatty acid fraction. Subsequently, the 1(st) reaction mixture was used for the 2(nd) enzymatic esterification without any separation process. The 2(nd) esterification was conducted for further SDA enrichment in a packed-bed reactor using Lipozyme RM IM from Rhizomucor miehei and the SDA content increased in a very short residence time. Ethanol was selected as an appropriate alcohol to react as an acyl receptor, and the other conditions for SDA enrichment were optimized at 20 degC of temperature, and 1:4 of molar ratio (i.e., fatty acid to ethanol). Under these conditions, 51.6% of SDA was obtained in the fatty acid fraction after a residence time of 15 min. Finally, highly purified SDA (purity, >99%) was obtained by prep-HPLC using the SDA-rich fraction obtained from the two-step lipase-catalyzed esterification.
Title: Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase Kim IH, Park YK, Nishiwaki H, Hammock BD, Nishi K Ref: Bioorganic & Medicinal Chemistry, 23:7199, 2015 : PubMed
Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore.
N,N'-disubstituted urea-based soluble epoxide hydrolase (sEH) inhibitors are promising therapeutics for hypertension, inflammation, and pain in multiple animal models. The drug absorption and pharmacological efficacy of these inhibitors have been reported extensively. However, the drug metabolism of these inhibitors is not well described. Here we reported the metabolic profile and associated biochemical studies of an N-adamantyl urea-based sEH inhibitor 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) in vitro and in vivo. The metabolites of AEPU were identified by interpretation of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or NMR. In vitro, AEPU had three major positions for phase I metabolism including oxidations on the adamantyl moiety, urea nitrogen atoms, and cleavage of the polyethylene glycol chain. In a rodent model, the metabolites from the hydroxylation on the adamantyl group and nitrogen atom were existed in blood while the metabolites from cleavage of polyethylene glycol chain were not found in urine. The major metabolite found in rodent urine was 3-(3-adamantyl-ureido)-propanoic acid, a presumably from cleavage and oxidation of the polyethylene glycol moiety. All the metabolites found were active but less potent than AEPU at inhibiting human sEH. Furthermore, cytochrome P450 (CYP) 3A4 was found to be a major enzyme mediating AEPU metabolism. In conclusion, the metabolism of AEPU resulted from oxidation by CYP could be shared with other N-adamantyl-urea-based compounds. These findings suggest possible therapeutic roles for AEPU and new strategies for drug design in this series of possible drugs.
Title: Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase Kim IH, Lee IH, Nishiwaki H, Hammock BD, Nishi K Ref: Bioorganic & Medicinal Chemistry, 22:1163, 2014 : PubMed
We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.
Title: Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil Choi JH, Kim BH, Hong SI, Kim CT, Kim CJ, Kim Y, Kim IH Ref: J Sci Food Agric, 92:870, 2012 : PubMed
BACKGROUND: The purpose of this study was to produce triacylglycerols (TAGs) enriched in pinolenic acid (PLA) at the sn-2 position using the principle of acyl migration, from the pine nut oil containing PLA esterified exclusively at the sn-3 position. RESULTS: Two types of lipase-catalysed reactions, i.e. redistribution and reesterification of fatty acids, were successively performed using seven commercially available lipases as biocatalysts. Of the lipases tested, Novozym 435 and Lipozyme TL IM were effective biocatalysts for positioning PLA at the sn-2 location. These biocatalysts were selected for further evaluation of the effects of reaction parameters, such as temperature and water content on the migration of PLA residues to the sn-2 position and TAG content. For both lipases, a significant decrease in TAG content was observed after the lipase-catalysed redistribution of fatty acids for both lipases. The reduced TAG content could be enhanced up to approx. 92%, through lipase-catalysed re-esterification of the hydrolysed fatty acids under vacuum. CONCLUSION: TAG enriched in PLA at the sn-2 position was synthesised from pine nut oil via lipase-catalysed redistribution and re-esterification of fatty acid residues using Lipozyme TL IM and Novozym 435 as biocatalysts.
Adamantyl ureas were previously identified as a group of compounds active against Mycobacterium tuberculosis in culture with minimum inhibitor concentrations (MICs) below 0.1 mug/ml. These compounds have been shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate. They also inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases. However, active compounds to date have high cLogP's and are poorly soluble, leading to low bioavailability and thus limiting any therapeutic application. In this study, a library of 1600 ureas (mostly adamantyl ureas), which were synthesized for the purpose of increasing the bioavailability of inhibitors of hsEH, was screened for activity against M. tuberculosis. 1-Adamantyl-3-phenyl ureas with a polar para substituent were found to retain moderate activity against M. tuberculosis and one of these compounds was shown to be present in serum after oral administration to mice. However, neither it, nor a closely related analog, reduced M. tuberculosis infection in mice. No correlation between in vitro potency against M. tuberculosis and the hsEH inhibition were found supporting the concept that activity against hsEH and M. tuberculosis can be separated. Also there was a lack of correlation with cLogP and inhibition of the growth of M. tuberculosis. Finally, members of two classes of adamantyl ureas that contained polar components to increase their bioavailability, but lacked efficacy against growing M. tuberculosis, were found to taken up by the bacterium as effectively as a highly active apolar urea suggesting that these modifications to increase bioavailability affected the interaction of the urea against its target rather than making them unable to enter the bacterium.
Lipase-catalyzed interesterification of high oleic sunflower oil and fully hydrogenated soybean oil (70 : 30, wt/ wt) was carried out in a packed bed reactor using an immobilized lipase from Thermomyces lanuginosus (Lipozyme TL IM) and the effect of a stepwise temperature protocol involving the 2 different temperatures, 60 and 70 degreeC, was investigated. The melting point of a fat that was incubated at 70 degreeC for 9 min was 57 degreeC, which suggested that it should be to employ a lower reaction temperature of 60 degreeC, after the first 9 min of the reaction. There were no significant differences (P < 0.05) in the conversion degree, triacylglycerol profile, and solid fat content between a constant temperature protocol (70 degreeC) and a stepwise temperature protocol (a combination of 70 and 60 degreeC). After 50 cycles, the overall residual activities of enzymes employed in stepwise temperature protocol were significantly (P < 0.05) higher than those of enzymes employed in constant temperature protocol.
Title: Structure-activity relationships of cycloalkylamide derivatives as inhibitors of the soluble epoxide hydrolase Kim IH, Park YK, Hammock BD, Nishi K Ref: Journal of Medicinal Chemistry, 54:1752, 2011 : PubMed
Structure-activity relationships of cycloalkylamide compounds as inhibitors of human sEH were investigated. When the left side of amide function was modified by a variety of cycloalkanes, at least a C6 like cyclohexane was necessary to yield reasonable inhibition potency on the target enzyme. In compounds with a smaller cycloalkane or with a polar group on the left side of amide function, no inhibition was observed. On the other hand, increased hydrophobicity dramatically improved inhibition potency. Especially, a tetrahydronaphthalene (20) effectively increased the potency. When a series of alkyl or aryl derivatives of cycloalkylamide were investigated to continuously optimize the right side of the amide pharmacophore, a benzyl moiety functionalized with a polar group produced highly potent inhibition. A nonsubstituted benzyl, alkyl, aryl, or biaryl structure present on the right side of the cycloalkylamide function induced a big decrease in inhibition potency. Also, the resulting potent cycloalkylamide (32) showed reasonable physical properties.
Title: Production of a novel cold-active lipase from Pichia lynferdii Y-7723 Kim HR, Kim IH, Hou CT, Kwon KI, Shin BS Ref: Journal of Agricultural and Food Chemistry, 58:1322, 2010 : PubMed
Lipase (triacylglycerol acylhydrolases, E.C. 3.1.1.3) is one of the most important enzymes applied to a broad range of industrial application fields. Especially, lipases with abnormal functionality such as thermostability and alkaline, acidic, and cold activities gain special attention because of their applicability in the restricted reaction conditions. In this study, 16 yeast strains prescreened for lipase induction were investigated for their actual lipase production, and we found a novel cold-active lipase produced from Pichia lynferdii Y-7723. The activity of lipase Y-7723 was retained by 74 and 70% at 20 and 10 degrees C, respectively, as compared to the maximum value at 35 degrees C. On the basis of an optimization study, the optimal lipase productivity was obtained at 96 h of incubation with 3% oil substrate in a medium composed of sucrose as a carbon source at pH 7.0. Among carbon sources tested, sucrose showed almost twice as high of a lipase production (184%) as the control, while the cell growth was similar (105%). Yeast extract and ammonium salts were effective as individual nitrogen sources for lipase production. This study demonstrated that the cold activity of lipase Y-7723 at 10 degrees C was highest among the cold-active lipases reported so far.
Epoxyeicosatrienoic acids that have anti-hypertensive and anti-inflammatory properties are mainly metabolized by soluble epoxide hydrolase (sEH, EC 3.3.2.3). Therefore, sEH has emerged as a therapeutic target for treating various cardiovascular diseases and inflammatory pain. N,N'-Disubstituted ureas are potent sEH inhibitors in vitro. However, in vivo usage of early sEH inhibitors has been limited by their low bioavailability and poor physiochemical properties. Therefore, a group of highly potent compounds with more drug-like physiochemical properties were evaluated by monitoring their plasma profiles in dogs treated orally with sEH inhibitors. Urea compounds with an adamantyl or a 4-trifluoromethoxyphenyl group on one side and a piperidyl or a cyclohexyl ether group on the other side of the urea function showed pharmacokinetic profiles with high plasma concentrations and long half lives. In particular, the inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) not only is very potent with good physiochemical properties, but also shows high oral bioavailability for doses ranging from 0.01 to 1mg/kg. This compound is also very potent against the sEH of several mammals, suggesting that t-AUCB will be an excellent tool to evaluate the biology of sEH in multiple animal models. Such compounds may also be a valuable lead for the development of veterinary therapeutics.
Title: Lipase-catalyzed acidolysis of olive oil with capric acid: effect of water activity on incorporation and acyl migration Oh JE, Lee KW, Park HK, Kim JY, Kwon KI, Kim JW, Kim HR, Kim IH Ref: Journal of Agricultural and Food Chemistry, 57:9280, 2009 : PubMed
Structured lipids were synthesized by acidolysis of olive oil and capric acid with an immobilized lipase (Lipozyme TL IM) from Thermomyces lanuginosus. The acidolysis reaction was carried out by incubating a 1:3 molar ratio of olive oil and capric acid under solvent-free reaction systems at 50 degrees C. The effect of water activity on the incorporation of capric acid was investigated, and the tested water activity range was between 0.22 and 0.80. Capric acid incorporation into triacylglycerols of the olive oil increased as the water activity increased, but the degree of acyl migration also increased. Also, the degree of acyl migration of modified olive oils with a similar degree of incorporation was investigated. High degrees of acyl migration occurred at water activities of 0.22 and 0.32 for the degree of incorporation of ca. 50 mol %. Only 8 h of reaction time was required to achieve incorporation of ca. 50 mol % at a water activity of 0.80, and the lowest acyl migration occurred at the same water activity. These results suggest that acyl migration can be efficiently minimized by a shorter reaction time at higher water activity.
Epoxyeicosatrienoic acids (EETs) are derived from cytochrome P450-catalyzed epoxygenation of arachidonic acid and have emerged as important mediators of numerous biological effects. The major elimination pathway for EETs is through soluble epoxide hydrolase (sEH)-catalyzed metabolism to dihydroxyeicosatrienoic acids (DHETs). Based on previous studies showing that EETs have anti-inflammatory effects, we hypothesized that chronic inhibition of sEH would attenuate a lipopolysaccharide (LPS)-induced inflammatory response in vivo. Continuous dosing of the sEH inhibitors 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA), a polyethylene glycol ester of AUDA, and 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)-pentyl)urea resulted in robust exposure to the inhibitor and target engagement, as evidenced by significant increases in plasma EET/DHET ratios following 6 days of inhibitor treatment. However, sEH inhibitor treatment was not associated with an attenuation of LPS-induced inflammatory gene expression in the liver, and AUDA did not protect from LPS-induced neutrophil infiltration. Furthermore, Ephx2-/-mice that lack sEH expression and have significantly increased plasma EET/DHET ratios were not protected from LPS-induced inflammatory gene expression or neutrophil accumulation in the liver. LPS did have an effect on sEH expression and function, as evident from a significant down-regulation of Ephx2 mRNA and a significant shift in plasma EET/DHET ratios 4 h after LPS treatment. In conclusion, there was no evidence that increasing EET levels in vivo could modulate an LPS-induced inflammatory response in the liver. However, LPS did have significant effects on plasma eicosanoid levels and hepatic Ephx2 expression, suggesting that in vivo EET levels are modulated in response to an inflammatory signal.
To determine whether sEH inhibitors influence atherosclerotic lesion formation, we used an established murine model of accelerated atherogenesis, ApoE knockout (-/-) mice. The sEH inhibitor, 1-adamantan-3-(5-(2-(2-ethylethoxy)ethoxy)pentyl)urea (AEPU) was delivered in drinking water. All animals were fed an atherogenic diet while simultaneously infused with angiotensin II by osmotic minipump to induce atherosclerosis. In AEPU-treated animals, there was a 53% reduction in atherosclerotic lesions in the descending aortae as compared to control aortae. AEPU and its major metabolites were detected in the plasma of animals which received it. As expected from the inhibition of sEH, a significant increase in linoleic and arachidonic acid epoxides, as well as an increase in individual 11,12-EET/DHET and 14,15-EET/DHET ratios, were observed. The reduction in atherosclerotic lesion area was inversely correlated with 11,12- and 14,15- EET/DHET ratios, suggesting that the reduction corresponds to the inhibition of sEH. Our data suggest that orally-available sEH inhibitors may be useful in the treatment of patients with atherosclerotic cardiovascular disease.
Title: 1,3-disubstituted ureas functionalized with ether groups are potent inhibitors of the soluble epoxide hydrolase with improved pharmacokinetic properties Kim IH, Tsai HJ, Nishi K, Kasagami T, Morisseau C, Hammock BD Ref: Journal of Medicinal Chemistry, 50:5217, 2007 : PubMed
Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokinetic properties. The structure-activity relationship studies showed that a hydrophobic linker between the urea group and the ether function is necessary to keep their potency. In addition, urea-ether inhibitors having a polar group such as diethylene glycol or morpholine significantly improved their physical properties and metabolic stability without any loss of inhibitory potency. Furthermore, improved pharmacokinetic properties in murine and canine models were obtained with the resulting inhibitors. These findings will facilitate the usage of sEH inhibitors in animal models of hypertension and inflammation.
The soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in blood pressure regulation and vascular inflammation. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA, 1) is a very active inhibitor of sEH both in vitro and in vivo. However, its relatively high melting point and limited solubility in either water or oil-based solvents leads to difficulties in formulating the compound and often results in poor in vivo availability. We investigated the effect of derivatization of the acid functional group of inhibitor 1 on the inhibition potencies, physical properties, and pharmacokinetic properties. For human sEH, similar inhibition potency was obtained when the acid of compound 1 was modified to esters (2-15). The resulting compounds exhibited improved physical properties (23-66 degrees C lower melting point and 5-fold better solubility in oil). Pharmacokinetic studies showed that the esters possess improved oral bioavailability in mice. On the other hand, amide derivatives of AUDA 1 did not show significant improvement in inhibition potencies or physical properties (higher melting points and lower solubility). The esterification of 1 results in compounds that are easier to formulate in animal food and in triglycerides for gavage and other routes of administration, making it easier to study the biological effects of sEH inhibition in vivo.
Title: New finding and optimal production of a novel extracellular alkaline lipase from Yarrowia lipolytica NRRL Y-2178 Lee GH, Bae JH, Suh MJ, Kim IH, Hou CT, Kim HR Ref: J Microbiol Biotechnol, 17:1054, 2007 : PubMed
Lipases are industrially useful versatile enzymes that catalyze numerous different reactions including hydrolysis of triglycerides, transesterification, and chiral synthesis of esters under natural conditions. Although lipases from various sources have been widely used in industrial applications, such as in food, chemical, pharmaceutical, and detergent industries, there are still substantial current interests in developing new microbial lipases, specifically those functioning in abnormal conditions. We screened 17 lipase-producing yeast strains, which were prescreened for substrate specificity of lipase from more than 500 yeast strains from the Agricultural Research Service Culture Collection (Peoria, IL, U.S.A.), and selected Yarrowia lipolytica NRRL Y-2178 as a best lipase producer. This report presents new finding and optimal production of a novel extracellular alkaline lipase from Y. lipolytica NRRL Y-2178. Optimal c ulture conditions f orlipase production by Y. lipolytica NRRL Y-2178 were 72 h incubation time, 27.5 degrees C, pH 9.0. Glycerol and glucose were efficiently used as the most efficient carbon sources, and a combination of yeast extract and peptone was a good nitrogen source for lipase production by Y. lipolytica NRRL Y-2178. These results suggested that Y. lipolytica NRRL Y-2178 showsgood industrial potential as a new alkaline lipase producer.
Title: Characterization of pyrethroid hydrolysis by the human liver carboxylesterases hCE-1 and hCE-2 Nishi K, Huang H, Kamita SG, Kim IH, Morisseau C, Hammock BD Ref: Archives of Biochemistry & Biophysics, 445:115, 2006 : PubMed
Carboxylesterases hydrolyze a large array of endogenous and exogenous ester-containing compounds, including pyrethroid insecticides. Herein, we report the specific activities and kinetic parameters of human carboxylesterase (hCE)-1 and hCE-2 using authentic pyrethroids and pyrethroid-like, fluorescent surrogates. Both hCE-1 and hCE-2 hydrolyzed type I and II pyrethroids with strong stereoselectivity. For example, the trans-isomers of permethrin and cypermethrin were hydrolyzed much faster than corresponding cis-counterparts by both enzymes. Kinetic values of hCE-1 and hCE-2 were determined using cypermethrin and 11 stereoisomers of the pyrethroid-like, fluorescent surrogates. K(m) values for the authentic pyrethroids and fluorescent surrogates were in general lower than those for other ester-containing substrates of hCEs. The pyrethroid-like, fluorescent surrogates were hydrolyzed at rates similar to the authentic pyrethroids by both enzymes, suggesting the potential of these compounds as tools for high throughput screening of esterases that hydrolyze pyrethroids.
Title: Substituted adamantyl-urea inhibitors of the soluble epoxide hydrolase dilate mesenteric resistance vessels Olearczyk JJ, Field MB, Kim IH, Morisseau C, Hammock BD, Imig JD Ref: Journal of Pharmacology & Experimental Therapeutics, 318:1307, 2006 : PubMed
The epoxyeicosatrienoic acids (EETs) have been identified as endothelium-derived hyperpolarizing factors. Metabolism of the EETs to the dihydroxyeicosatrienoic acids is catalyzed by soluble epoxide hydrolase (sEH). Administration of urea-based sEH inhibitors provides protection from hypertension-induced renal injury at least in part by lowering blood pressure. Here, we investigated the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their action on the vasculature. Mesenteric resistance arteries were isolated from Sprague-Dawley rats, pressurized, and constricted with the thromboxane A2 agonist U46619 (9,11-dideoxy-11,9-epoxymethano-prostaglandin F2alpha). Mesenteric arteries were then incubated with increasing concentrations of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). AUDA resulted in a concentration-dependent relaxation of mesenteric arteries, with 10 microM resulting in a 48 +/- 7% relaxation. Chain-shortened analogs of AUDA had an attenuated vasodilatory response. Interestingly, at 10 microM, the sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexylureido)dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active, resulting in a 25 +/- 8%, 10 +/- 9%, and -8 +/- 3% relaxation, respectively. Treatment of mesenteric arteries with tetraethylammonium, iberiotoxin, ouabain, or glibenclamide did not alter AUDA-induced relaxation. The AUDA-induced relaxation was completely inhibited when constricted with KCl. In separate experiments, denuding mesenteric resistance vessels did not alter AUDA-induced relaxation. Taken together, these data demonstrate that adamantyl-urea inhibitors have unique dilator actions on vascular smooth muscle compared with other sEH inhibitors and that these dilator actions depend on the adamantyl group and carbon chain length.
Combination therapies have long been used to treat inflammation while reducing side effects. The present study was designed to evaluate the therapeutic potential of combination treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and previously undescribed soluble epoxide hydrolase inhibitors (sEHIs) in lipopolysaccharide (LPS)-challenged mice. NSAIDs inhibit cyclooxygenase (COX) enzymes and thereby decrease production of metabolites that lead to pain and inflammation. The sEHIs, such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), stabilize anti-inflammatory epoxy-eicosatrienoic acids, which indirectly reduce the expression of COX-2 protein. Here we demonstrate that the combination therapy of NSAIDs and sEHIs produces significantly beneficial effects that are additive for alleviating pain and enhanced effects in reducing COX-2 protein expression and shifting oxylipin metabolomic profiles. When administered alone, AUDA-BE decreased protein expression of COX-2 to 73 +/- 6% of control mice treated with LPS only without altering COX-1 expression and decreased PGE(2) levels to 52 +/- 8% compared with LPS-treated mice not receiving any therapeutic intervention. When AUDA-BE was used in combination with low doses of indomethacin, celecoxib, or rofecoxib, PGE(2) concentrations dropped to 51 +/- 7, 84 +/- 9, and 91 +/- 8%, respectively, versus LPS control, without disrupting prostacyclin and thromboxane levels. These data suggest that these drug combinations (NSAIDs and sEHIs) produce a valuable beneficial analgesic and anti-inflammatory effect while prospectively decreasing side effects such as cardiovascular toxicity.
The soluble epoxide hydrolase (sEH) metabolizes vasodilatory epoxyeicosatrienoic acids (EETs) to their di-hydroxy derivatives. We hypothesized that the metabolism of EETs by the sEH contributes to angiotensin II-induced hypertension and tested the effects of a water-soluble sEH inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) on blood pressure. AUDA (130 microg/mL in drinking water) did not affect blood pressure in normotensive animals but markedly lowered it in mice with angiotensin II-induced hypertension (1 mg/kg per day). The effect of AUDA was accompanied by an increase in urinary salt and water excretion. Intravenous application of AUDA (8 mg/kg) acutely lowered blood pressure and heart rate in animals with angiotensin II-induced hypertension but failed to affect blood pressure in animals with phenylephrine-induced hypertension (29 mg/kg per day). AUDA (0.1 micromol/L) selectively lowered vascular resistance in an isolated perfused kidney preparation from angiotensin II-pretreated mice but not from control mice. In the perfused hind limb and in isolated carotid arteries from angiotensin II-treated mice, AUDA was without effect. The omega-hydroxylase inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide, which attenuates formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, decreased tone in carotid arteries from angiotensin II-treated but not from control mice. These data demonstrate that the decrease in blood pressure observed after sEH inhibition in angiotensin II-induced hypertension can be attributed to an initial reduction in heart rate followed by pressure diuresis resulting from increased perfusion of the kidney. Direct vasodilatation of resistance arteries in skeletal muscles does not appear to contribute to the antihypertensive effects of sEH inhibition in mice.
Soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in the regulation of blood pressure and inflammation. 1,3-Disubstituted ureas with a polar group located on the fifth atom from the carbonyl group of urea function are active inhibitors of sEH both in vitro and in vivo. However, their limited solubility in water and relatively high melting point lead to difficulties in formulating the compounds and poor in vivo efficacy. To improve these physical properties, the effect of structural modification of the urea pharmacophore on the inhibition potencies, water solubilities, octanol/water partition coefficients (log P), and melting points of a series of compounds was evaluated. For murine sEH, no loss of inhibition potency was observed when the urea pharmacophore was modified to an amide function, while for human sEH 2.5-fold decreased inhibition was obtained in the amide compounds. In addition, a NH group on the right side of carbonyl group of the amide pharmacophore substituted with an adamantyl group (such as compound 14) and a methylene carbon present between the adamantyl and amide groups were essential to produce potent inhibition of sEH. The resulting amide inhibitors have 10-30-fold better solubility and lower melting point than the corresponding urea compounds. These findings will facilitate synthesis of sEH inhibitors that are easier to formulate and more bioavailable.
As of 2004, >73 million people were prescribed antiinflammatory medication. Despite the extensive number of current products, many people still suffer from their diseases or the pharmacological properties (side effects) of the medications. Therefore, developing therapeutic strategies to treat inflammation remains an important endeavor. Here, we demonstrate that the soluble epoxide hydrolase (sEH) is a key pharmacologic target for treating acute systemic inflammation. Lipopolysaccharide-induced mortality, systemic hypotension, and histologically evaluated tissue injury were substantially diminished by administration of urea-based, small-molecule inhibitors of sEH to C57BL/6 mice. Moreover, sEH inhibitors decreased plasma levels of proinflammatory cytokines and nitric oxide metabolites while promoting the formation of lipoxins, thus supporting inflammatory resolution. These data suggest that sEH inhibitors have therapeutic efficacy in the treatment and management of acute inflammatory diseases.
Title: Design, synthesis, and biological activity of 1,3-disubstituted ureas as potent inhibitors of the soluble epoxide hydrolase of increased water solubility Kim IH, Morisseau C, Watanabe T, Hammock BD Ref: Journal of Medicinal Chemistry, 47:2110, 2004 : PubMed
The soluble epoxide hydrolase (sEH) is involved in the metabolism of endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas are potent inhibitors of sEH that are active both in vitro and in vivo. However, their poor solubility in either water or lipid reduces their in vivo efficacy and makes them difficult to formulate. To improve these physical properties, the effect of incorporating polar functional groups into one of the alkyl chains was evaluated on their inhibitor potencies, water solubility, octanol/water partition coefficients (log P), and melting points. No loss of inhibition potency was observed when a polar functional group was incorporated at least five atoms ( approximately 7.5 A) from the central urea carbonyl. In addition, the presence of a polar group at least 11 atoms away from the urea carbonyl group for the mouse and human sEHs, respectively, did not alter the inhibitor potency. The resulting compounds have better water solubility and generally lower log P values and melting points than nonfunctionalized liphophilic ureas. These properties will make the compounds more bioavailable and more soluble in either water- or oil-based formulations.
Epoxyeicosatrienoic acids (EET) have antihypertensive and anti-inflammatory properties and play a role in the maintenance of renal vascular function. A novel approach to increase EET levels is to inhibit epoxide hydrolase enzymes that are responsible for conversion of biologically active EET to dihydroxyeicosatrienoic acids (DHET). We hypothesized that soluble epoxide hydrolase (SEH) inhibition would improve renal vascular function and ameliorate hypertension induced renal damage. Chronic administration of the specific SEH inhibitor 1-cyclohexyl-3-dodecylurea (CDU, 3 mg/d) for 10 d lowered BP in angiotensin hypertensive rats. The contribution of renal vascular SEH to afferent arteriolar function in angiotensin hypertension was also assessed. SEH protein expression was increased in renal microvessels from hypertensive rats. Although CDU did not change afferent arteriolar responsiveness to angiotensin in normotensive animals, CDU treatment significantly attenuated afferent arteriolar diameter responses to angiotensin in hypertensive kidneys from 51% +/- 8% to 28% +/- 7%. Protection of the renal vasculature and glomerulus during chronic CDU administration was demonstrated by histology. Urinary albumin excretion, an index of renal damage, was also lower in CDU-treated hypertensive rats. These data demonstrate that SEH inhibition has antihypertensive and renal vascular protective effects in angiotensin hypertension and suggests that SEH inhibitors may be a useful therapeutic intervention for cardiovascular diseases.
