Quantification of soluble epoxide hydrolase inhibitors in experimental and clinical samples using the nanobody-based ELISA

To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.

IMPROVEMENT OF WET-CREASE RECOVERY PROPERTIES OF SILK FABRICS TREATED WITH EPOXIDES

Silk fabrics is finished with epoxide TDEA or TDEB. By the determination of washing fastness, DSC, IR spectrum and amino acid contents tests, TDEA or TDEB is confirmed to react with silk fibroin. By means of tests of silk fibroin swelling and solubility in some solvents, crosslinking reactions between TDEA or TDEB and silk fibroin arc further proved, which is obviously responsible to the increase of wet-crease recovery of silk fabrics finished with epoxide TDEA or TDEB.

（Salen）Ti（Ⅳ）-Catalyzed Asymmetric Ring-opening of meso Epoxides Using Dithiophosphorus Acid as the Nucleophile

The asymmetric ring-opening of epoxides with dithiophosphorus acids catalyzed by a （salen）Ti（Ⅳ） complex formed in situ from the reaction of Ti（OPr-i）4 and the chiral Schiff base derived from （1R,2R）-（＋）-diaminocyclohexane was realized. The resulting products were obtained with low to good enantioselectivity （up to 73% ee）.

SYNTHESIS AND CHARACTERIZATION OF HYDROXYLATED FULLERENE EPOXIDE—AN INTERMEDIATE FOR FORMING FULLEROL

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Nuclephilic ring opening of epoxides promoted by multi-site phase-transfer catalyst:An efficient and eco-friendly route to synthesis of β-hydroxythiocyanate

A highly effective and mild protocol for ring opening of epoxides with NH4SCN in the presence of catalytic amount of a multi- site phase-transfer catalyst, α,α＇,α＂-N-hexakis（triethylammoniummethylene chloride）-melamine, is developed. A variety of ^-hydroxy thiocyanates as important intermediates in agricultural and pharmaceutical chemistry were obtained in high yields with excellent regioselectivity and in short reaction times. 2009 Ali Reza Kiasat. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Highly active double metal cyanide complexes: Effect of central metal and ligand on reaction of epoxide/CO2

Various novel double metal cyanide （DMC） catalysts were successfully prepared by modifying the central metal （M） and one of cyanide ion （CN-） in Zna[M（CN）b]c complex. Such modifications have significant impact on the catalytic efficiency as well as the polymer selectivity for the reaction of PO/CO2. Zn-Ni（Ⅱ） DMC is a potential catalyst for alternating copolymerization of PO/CO2,and DMC catalysts based on Zn3[Co（CN）5X]2 （X = Br^- and N3^-） exhibit moderate efficiency for the production of polycarbonates.This research presents the preliminary exploration of novel DMC complex via chemical modification of its central metal and ligand.

Decreased Expression of Vitamin K Epoxide Reductase Complex Subunit 1 in Kidney of Patients with Calcium Oxalate Urolithiasis

Urinary prothrombin fragment 1 （UPTFl） is a potent inhibitor of urinary stone formation. UPTF1 exerts such inhibitory effect by effective 7-carboxylation in which vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme, is involved. This study examined the correlation between VKORC1 expression and calcium oxalate urolithiasis. The renal cortex samples were obtained from patients undergoing nephrectomy and then divided into 3 groups： urolithiasis group, control group A [hydronephrosis-without-stone （HWS） group], control group B （normal control group）, The localization and expression of VKORC1 in renal tissues were determined by using immunohistochemistry, immunofluorescence microscopy, Western blotting and SYBR Green I real-time reverse-transcription PCR. The rapid amplification of cDNA ends （RACE） were conducted to obtain the 3＇- and 5＇-untranslated region （UTR） of VKORC1. The results showed that VKORC1 was located in the cytoplasm of renal tubular epithelial cells. The expression of VKORC1 in the uro- lithiasis group was significantly lower than that in the other two control groups （P〈0.05）. Moreover, the 3＇- and 5＇-UTR sequence of the VKORC1 gene was successfully cloned. No insertion or deletion was found in the 3＇- and 5＇-UTR. However, a 171-bp new base sequence was discovered in the up- stream of 5＇-UTR end in the urolithiasis group. It was concluded that the decreased expression of VKORC 1 may contribute to the development of calcium oxalate urolithiasis in the kidney.

Synthesis of Dimethyl Carbonate from CO_2, Methanol, and Epoxides Using Re(CO)_5Cl/K_2CO_3 as Catalyst System

Over the past few years, dimethyl carbonate （DMC） has been proven to be an efficient methylating, methoxylating, and methoxycarbonylating agent in organic syntheses, in which DMC is used to replace the toxic methyl halides, dimethyl sulfate or carbon monoxide, and so forth. Furthermore, DMC has been used in the syntheses of polycarbonates and polyurethane to develop the nonphosgene routes Therefore, DMC has become one of the most important compounds in the development of green synthetic chemistry.

SYNTHESIS, CHARACTERIZATION, METAL-UPTAKE BEHAVIOR OF BIS(BENZIMIDAZOLE) RESIN SUPPORTED ON EPOXIDE POLYMER

The new chelate resins, abbreviated as PNBMZs and PBBMZs based on epoxide polymer, were synthesized by polycondensation of N,N-diglycidyl-4-glycidyloxyaniline or 1,4-bis（2,3-epoxypropyl）benzene with the primary amine group of 1,3-bis（benzimidazol-2yl）propylamine （BBPAH）. The ion exchangers contain 2.71-3.23 mmol of the ligand contents per gram of the resin. Batch extraction capacities were determined for the metal chloride salts in buffer solutions in the pH range from -1 to 6.0. The chelate resins were very selective for Cu^2＋, Zn^2＋, Cd^2＋ in the presence of other divalent transition metal ions. The maximum uptake capacities of PNBMZ （synthetic molar ratio = 1：1.5） under non-competitive condition were found to be 0.94 mmol/g for Cu^2＋ at pH = 2, 1.3 mmol/g for Cd^2＋ at pH = 1 and 1.75 mmol/g for Zn^2＋ at pH = -1 respectively. While in the case of PBBMZ, it was 1.39 mmol/g for Cd〉 at pH = 1. The metal-uptake behaviors for both of them showed strong pH dependence, and their extraction capacities increase with decreasing pH. The uptake of Cu^2＋ by the resin PNBMZs at pH = 1 was found to be rather fast with t1/2 = 18 min. Metal-uptake experiments under competitive conditions also confirm that the chelate resins have a high selectivity for Cu^2＋, Zn^2＋, Cd^2＋ and the contrary pH dependence.

Influence of Silencing Soluble Epoxide Hydrolase with RNA Interference on Cardiomyocytes Apoptosis Induced By Doxorubicin

In order to investigate the influence of silencing soluble epoxide hydrolase（sEH） with double-stranded small interfering RNA（siRNA） on cardiomyocytes apoptosis induced by doxorubicin（DOX）,two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence（PCN） served as the negative control.Cardiomyocytes were divided into four groups：control group,DOX group,PCN＋DOX group,and EH-R＋DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R＋DOX group were significantly decreased as compared with other groups（P0.01）.As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased（P0.01）.However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R＋DOX group when compared with those in the DOX group and the PCN＋DOX group（P0.01 for each）.It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

Science Letters:EHPred: an SVM-based method for epoxide hydrolases recognition and classification

A two-layer method based on support vector machines (SVMs) has been developed to distinguish epoxide hydrolases (EHs) from other enzymes and to classify its subfamilies using its primary protein sequences. SVM classifiers were built using three different feature vectors extracted from the primary sequence of EHs: the amino acid composition (AAC), the dipeptide composition (DPC), and the pseudo-amino acid composition (PAAC). Validated by 5-fold cross tests, the first layer SVM clas- sifier can differentiate EHs and non-EHs with an accuracy of 94.2% and has a Matthew’s correlation coefficient (MCC) of 0.84. Using 2-fold cross validation, PAAC-based second layer SVM can further classify EH subfamilies with an overall accuracy of 90.7% and MCC of 0.87 as compared to AAC (80.0%) and DPC (84.9%). A program called EHPred has also been developed to assist readers to recognize EHs and to classify their subfamilies using primary protein sequences with greater accuracy.

A New Catalyst in the Crease Recovery Finishing of Silk Fabrics with Epoxide

The paper here intends to discuss silk crease recovery finishing behavior with ED GE (ethylene diglycidal ether) in the presence of a new catalyst WSH. The results show that ED GE reacts with silk fibroin and forms cross linkages through ether bond. The formation of cross linking was examined by FITR spectroscopy, soluble time in acid solution and changes of amino acids. Handle of treated silk with catalyst WSH is better than that of silk treated with the traditional catalysts such as thiosulfates and thiocyanates. Finishing technique, physical properties of the finished silk fabrics and evaluation of cross linkages are discussed.

Al(HSO_4)_3/silica gel as a novel catalytic system for the ring opening of epoxides with thiocyanate anion under solvent-free conditions

For the first time,metal hydrogen sulfates and phosphates/silica gel have been studied as efficient and powerful solid acid catalysts in the ring opening of epoxides with thiocyanate anion.The most significant result was obtained by Al(HSO4)3/SiO2which afforded the corresponding β-hydroxy thiocyanates under mild reaction conditions and in very short reaction times.The cheapness, availability of the catalyst,ease of procedure and work-up make this method attractive for the organic synthesis.

Hydrolysis of epoxides and aziridines catalyzed by polymer-supported quarternary ammonium bisulfate

Macroporous resin （D201）-supported quartemary ammonium bisulfate （D201-HSO4） was prepared and effectively used in catalyzing the hydrolysis of epoxides or aziridines under mild and non-metal conditions to give the corresponding 1,2-diols and β-amino alcohols in high yields. The catalyst was facilely prepared and recyclable.

DEVELOPMENT OF LIGNIN EPOXIDE-A POTENTIAL MATRIX OF RESIN MATRIX COMPOSITE

To develop new kinds of matrix resin of composite and utilize lignosulfonate in large scale and high value, a Kind of lignosulfonate was modified into epoxide in this paper. Two kinds of phenolized lignosulfonic acid and two kinds of lignin epoxides were prepared. The lignin epoxides and a kind of bisphenol - A epoxy resin were mixed respectively with a kind of liquid anhydride (MNA) to be measured by DSC technology. Both of these two kinds of epoxides can be cured by MNA, and curing exotherm of the liquid one is more than that of the solid one. Samples of the phenolized lignosulfonic acid and the epoxides were all analyzed by IR spectroscopy to find relations between them. It is suggested that the phenolation decide the type and the yield of the lignin epoxides.

Lewis Acidic Guanidinium Ionic Liquids: Novel, Efficient and Recyclable Catalysts for Aminolysis of Epoxides

Lewis acidic guanidinium ionic liquid（LAGIL） 2c was used as a novel, efficient and recyclable catalyst for aminolysis of epoxides under solvent-free and room temperature conditions, giving the corresponding β-amino alcohols with moderate to excellent regioselectivity（up to 91：9） in high yields（up to 97%）. In addition, LAGIL 2c was recycled three times without any loss of catalytic activity and selectivity to the product.

Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues(V195, V196, F218,N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH^(V196A/N226A/M332A) had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg^-1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining(R)-PGE of E. coliSpEH^(V196A/N226A/M332A)increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH^(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.

Study on the Intramolecular Cyclization through Openmg of Epoxide IV. Cyclization of N-Methyl-N-benzoylmethylene β-Methyl-α, β- epoxypropionamide in Aprotic and Protic Solvents

Intramolecular cyclization of β -methyl substituted epoxide under aprotics （e.g. LDA in benzene） and protic conditions （e.g. LiOH in MeOH/H2O）both gave a mixture of β-,γ-, δ- and ε-lactam but in different ratio.