Methyl Ester Conjugate Research Articles

Widely targeted metabolomics analysis of enriched secondary metabolites and determination of their corresponding antioxidant activities in Elaeagnus angustifolia var. orientalis (L.)Kuntze fruit juice enhanced by Bifidobacterium animalis subsp. Lactis HN-3 fermentation

Elaeagnus angustifolia var. orientalis (L.)Kuntze fruit contains a large number of naturally occurring molecules present as glycoside, methylated, and methyl ester conjugates, which should be hydolysed or transformed to become bioactive forms. For this purpose, Bifidobacterium animalis subsp. lactis HN-3 was selected to ferment Elaeagnus angustifolia var. orientalis (L.)Kuntze fruit juice (EOJ). After fermentation, the total phenolic content (TPC) and antioxidant capacity of the EOJ increased significantly compared to the non-fermented EOJ. Using widely-targeted metabolomics analysis, polyphenolic compounds involved in the flavonoid biosynthetic pathway were determined to be up-regulated in the fermented EOJ. In addition, the metabolites generated by 8 deglycosidation, 5 demethylation, 5 hydrogenation, and 28 other reactions were detected in higher concentrations in the fermented EOJ compared to the non-fermented EOJ. Interestingly, these up-regulated metabolites have higher antioxidant and other biological activities than their metabolic precursors, which provide a theoretical basis for the development of Bifidobacterium-fermented plant products with stronger functional activities.

Preparation of synthetic auxin-amino acid conjugates

Auxin amide conjugates are regulators of the most important auxin, indole-3-acetic acid (IAA), which is considered responsible for many important processes within the plants. Herein, amide conjugates of IAA were synthesized employing a simple and efficient coupling method with WSCI·HCl, a water-soluble condensing reagent, in the presence of 1-hydroxybenzotriazole. IAA conjugates with 10 amino acids along with their corresponding methyl esters were prepared in excellent yields, up to 95%, aiming to facilitate their identification in plant species. Eight IAA-amino acid methyl ester conjugates are characterized here for the first time.

Synthesis of N-methyl-bisindolylmaleimide amino acid methyl ester conjugates and cytotoxicity study

A novel series of N-methylbisindolylmaleimides derivatives bearing 2-acetamino acid moieties were synthesized. The cytotoxic activities of these compounds were tested in six tumor cell lines. The most potent compound 8d displayed cytotoxicity against six human tumor cell lines in the micromolar range.

Bile acid–amino acid ester conjugates: gelation, structural properties, and thermoreversible solid to solid phase transition

Design, synthesis, and gelation properties of three novel biocompatible bile acid–L-methionine methyl ester conjugates are presented. Two of the conjugates have been shown to undergo self-assembly leading to organogelation in certain aromatic solvents. The properties of these gels have been investigated by conventional methods typical for molecular gel studies along with 13C CPMAS NMR spectroscopic studies of the native gel. In addition, properties in solid and solution states for all three compounds have been investigated, and single crystal X-ray structures of all compounds determined. Furthermore, powder X-ray diffraction studies have revealed that compound 1 undergoes a dynamic and reversible conformational change in the solid state when cooling from ambient temperature to −150 °C. The powder X-ray diffraction data of the room-temperature conformer has been utilized to unambiguously determine the structure at room temperature.

Inhibitory and structural studies of novel coenzyme‐substrate analogs of human histidine decarboxylase

Histamine, a biogenic amine with important biological functions, is produced from histidine by histidine decarboxylase (HDC), a pyridoxal 5'-phosphate-dependent enzyme. HDC is thus a potential target to attenuate histamine production in certain pathological states. Targeting mammalian HDC with novel inhibitors and elucidating the structural basis of their specificity for HDC are challenging tasks, because the three-dimensional structure of mammalian HDC is still unknown. In the present study, we designed, synthesized, and tested potentially membrane-permeable pyridoxyl-substrate conjugates as inhibitors for human (h) HDC and modeled an active site of hHDC, which is compatible with the experimental data. The most potent inhibitory compound among nine tested structural variants was the pyridoxyl-histidine methyl ester conjugate (PHME), indicating that the binding site of hHDC does not tolerate groups other than the imidazole side chain of histidine. PHME inhibited 60% of the fraction of 12-O-tetradecanoylphorbol-13-acetate-induced newly synthesized HDC in human HMC-1 cells at 200 microM and was also inhibitory in cell extracts. The proposed model of hHDC, containing phosphopyridoxyl-histidine in the active site, revealed the binding specificity of HDC toward its substrate and the structure-activity relationship of the designed and investigated compounds.

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3- O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3- O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1 H and 13 C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3- O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125 I -labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20–4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.

Radioimmunoassay kit formulation and its validation for serum progesterone using progesterone radiotracer purified by gel filtration

Purification of the radioiodinated progesterone tyrosine methyl ester conjugate by gel filtration and the development, optimization and clinical validation of a direct radioimmunoassay (RIA) of progesterone using this radiotracer are described. High purity radiotracer is essential for the error free performance of any RIA. Progesterone 11α hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mixed anhydride method and this conjugate was then radioiodinated by the chloramine-T method. Purification of the radioiodinated product was carried out by gel filtration. About 12 batches of the radiotracer were prepared and purified. The purification by gel filtration gave reproducible elution pattern and purity. The radiotracer thus purified was found to have consistent quality as compared to that of any other purification methods. Non-specific binding of the radiotracer was found to be 95% as checked by paper electrophoresis. The stability (retention of the immunoreactivity) of the radiotracer was two to three months. No appreciable changes in the assay characteristics were observed during this period. The assay involved 3 hours incubation of progesterone antibody with individual standards or sample and radiotracer at room temperature. The optimized assay was then validated for internal and external quality control parameters. A RIA kit was then formulated with this radiotracer for estimation of progesterone in serum. The assay kit consisted of lyophilized individual standards ranging from 0.25 to 50 ng/ml. The clinical performance of the developed kit was compared with that of a commercial ELISA kit and a correlation of 0.94 was observed.

Development of radioimmunoassay

Therapeutic monitoring of theophylline can be accurately performed by radioimmunoassay (RIA). It is radioactive tracer as an essential reagent for the development of very sensitive RIA. Direct radiolabeling of theophylline with125I is very difficult due to the absence of appropriate functional groups. Hence carboxylic acid of theophylline was tagged to tyrosine methyl ester and then radiolabeled. The derivatives of theophylline, bearing a propionic acid and butyric acid side chains at seventh and eight position of theophylline, were synthesised and coupled to tyrosine methyl ester. Theophylline-tyrosine methyl ester conjugates were labeled with125I using chlora mine—T. Radiolabeled theophylline was purified by solvent extraction followed by thin layer chromatography. The purified radiolabeled compound were assessed for their radiochemical purity, specific activity and immunoreactivity. Stability studies of radiolabeled compounds were performed with different solvents at different temperatures. Theophylline serum samples analysed using developed and commercial kits showed the correlation coefficient of 0.961 (n=9).

Synthesis of N,N-didemethylzolpidem-N-{2-{3-(4-hydroxy-3-[125I] iodophenyl)}methyl propionate} for use in radioimmunoassay

The 125 I-derivative of zolpidem was prepared by iodination of a L-tyrosine methyl ester conjugate of a zolpidem precursor. The iodinated compound has been used as a tracer molecule in the development of a radioimmunoassay for zolpidem. It was purified by normal phase HPLC, in combination with gamma counting detection. The structure was confirmed by high resolution L-SIMS

Synthesis of a new 125I-labeled tyrosine methyl ester conjugate of adenylyl-(2′-5′)-adenylyl-(2′-5′)-[2′,3′-di-O-(2-carboxyethyl)ethylidene]adenosine

A novel 125I-labeled conjugate of 2′,5′-oligoadenylate trimer for radioimmuno assay of 2′,5′-oligoadenylates have been obtained by the reaction of adenylyl-(2′-5′)-adenylyl-(2′-5′)-[2′,3′-di-O-(2-carboxyethyl)ethylidene]adenosine ( 1) with tyrosine methyl ester followed by iodination of intermediate 2 with 125I in the presence of chloramine-T. 125I-labeled conjugate 3 was isolated by column chromatography on a TSK-gel HW-40 in 73% yield. Specific activity for the synthesized conjugate was approximately 1760 Ci/mmol.

Radioimmunological and Chromatographic Properties of Tyrosine Methyl Ester Conjugates with Stereoisomeric Steroid Carboxy Derivatives

Pure 3Z (syn) and 3E (anti) stereoisomers of testosterone 3-[O-(2-carboxyethyl)]oxime were synthesized, separated by HPLC or TLC, and used for preparation of tyrosine methyl ester (TME) conjugates by using mixed anhydride or carbodiimide-N-hydroxysuccinimide methods. While the latter method provided more than 96% of product with retained configuration, the mixed anhydride method yielded a mixture containing 26-40% of the opposite stereoisomer. The stereoisomers were used as model compounds, to which the other steroid TMEs and the corresponding radioiodinated products could be aligned according to their chromatographic properties. The TME conjugates of 3-(O-carboxymethyl)oximes of seven 4-en-3-oxo steroids were further prepared by carbodiimide-N-hydroxysuccinimide method. With exception of cortisol, the stereoisomeric (Z and E) radioiodinated TME conjugates could be separated by TLC. In addition, the conjugates with TME and consequently radioiodinated tracers were synthesized from hemisuccinates of cortisol and its 11α-isomer, via 11β- and 11α-hydroxy group. The radioiodinated conjugates were tested as radioligands with rabbit polyclonal antisera raised by using position-homologous conjugates of the respective steroid carboxy derivatives with bovine serum albumin as immunogens. With the exception of 11-deoxycorticosterone, the stereoisomeric Z and E radioiodinated TMEs did not differ in their binding properties. In the case of isomeric cortisol tracers conjugated through position 11 the antisera recognized only the sterically homologous radioligands, but the specificity of the system was poor.

Aprikalim: Radioimmunoassay and Pharmacokinetic Studies in Mouse, Monkey, and Dog

A stereoselective and specific radioimmunoassay (RIA) was developed for aprikalim (RP 52891), a novel potassium channel opener. Antibodies were produced in rabbits immunized with the pure levorotatory enantiomer (1R.2R) of the hapten derivative bearing an acid function at the end of the lateral chain and conjugated to bovine serum albumin. In displacement studies with the enantiomerically pure radioligand (radioiodinated tyrosine methyl ester conjugate of the hapten derivative), the opposite enantiomer showed only 0.1 % crossreaction. Negligible binding occurred when analogues or metabolites of aprikalim were tested for cross-reactivity. The detection limit was 0. 25 ng/mL (9.31 X 10-10 M) in a 20-nL plasma sample. The assay was used successfully to determine aprikalim pharmacokinetics in mice, monkeys, and dogs. Beagle dogs were given a 10 jug/kg intravenous (iv) bolus dose or 10 /tg/kg iv bolus followed by 0.1 fig! kg/min infused over 30min (nonhypotensive doses which reduce myocardial infarct size significantly). The plasma concentrations declined monoexponentially with a mean overall elimination half-life of 1.53h and a mean plasma clearance of 52mL/min (5.1 mL/min/ kg). A slow-release oral formulation produced a significant delay in the rate of absorption, a 4-fold decrease in the peak plasma level, and a 2-fold decrease in apparent oral bioavailability relative to that observed for an oral solution. A comparison of aprikalim pharmacokinetic parameters in mouse, monkey, and dog revealed great similarity in disposition characteristics in these species.

4-fluorobenzylamine and phenylalanine methyl ester conjugates OF 2-nitroimidazole: Evaluation as hypoxic cell radiosensitizers

We have synthesized two 2-nitroimidazole derivatives and evaluated their hypoxic radiosensitization properties. The first, a 4-fluorobenzylamine conjugate of 2-nitroimidazole (PK-110), was designed so that it could also be labeled with the F-18 and used for positron emission tomographic imaging of hypoxia. The second, the L-phenylalanine methyl ester conjugate of 2-nitroimidazole (PK-130), was designed in an attempt to exploit amino acid transport channels to enhance drug transport into the tumor. The effects of these drugs (and SR-2508, for comparison) in vitro on the aerobic and hypoxic radiosensitivity of Chinese hamster V79 cells were evaluated using clonogenic assays. PK-130 and PK-110 at 0.1 and 1.0 mM were more efficient hypoxic cell radiosensitizers than SR-2508 at the same concentrations. PK-130 and PK-110 at 0.1 mM yielded comparable SER's to that obtained with 1.0 mM SR-2508. Marginal aerobic radiosensitization was observed for 1.0 mM treatment with PK-130 and PK-110, however, no aerobic radiosensitization was observed at 0.1 mM. Glutathione (GSH) depletion (< 5% of control levels) by L-buthionine sulfoximine (BSO) further enhanced the SER for both PK-130 and PK-110 at 0.1 mM to 3.2 ± 0.63 and 2.4 ± 0.16, respectively. The results of this study encourage the in vivo tumor radiosensitization evaluation of PK-130 and PK-110.

Purification and characterization of an NAD +-dependent dehydrogenase that catalyzes the oxidation of thromboxane B 2 at C-11 from porcine liver. Development and application of 11-dehydro-thromboxane B 2 radioimmunoassay to enzyme assay

11-Dehydro-thromboxane B 2 has been identified as a major metabolite of infused as well as endogenous thromboxane B 2 in mammalian plasma and urine. This metabolite is derived from thromboxane B 2 by enzymatic oxidation at C-11 catalyzed by 11-hydroxytrhomboxane B 2 dehydrogenase. A radioimmunoassay for 11-dehydro-thromboxane B 2 has been developed and used for enzyme assay, purification and characterization. Antibodies were generated against 11-dehydrothromboxane B 2 conjugated to bovine thyroglobulin. Labeled marker was prepared by radioiodinating 11-dehydrothromboxane B 2-tyrosine methyl ester conjugate. A sensitive radioimmunoassay capable of detecting 10 pg of 11-dehydro-thromboxane B 2 per assay tube was developed. The antibodies showed minimal crossreaction with thromboxane B 2 (0.03%), prostaglandin D 2 (2.76%) and other eicosanoids (<0.03%). The enzyme activity was determined by assaying NAD +-dependent formation of immunoreactive 11-dehydro-thromboxane B 2 from thromboxane B 2. The enzyme was found to be enriched in liver although significant activity was also detected in gastrointestinal tract and kidney in pig. The enzyme was purified from porcine liver cytosol to apparent homogeneity using conventional and affinity chromatography. The purfied enzyme exhibited coenzyme specificity for NAD + and used thromboxane B 2 as a substrate. The enzyme also catalyzes NADH-dependent reduction of 11-dehydro-thromboxane B 2 to thromboxane B 2 indicating the reversibility of the enzyme catalyzed reaction. The apparent K m values for thromboxane B 2, 11-dehydrothromboxane B 2 and NAD + are 8.1, 8.0 and 23 μM, respectively. Subunit M r was shown to be 55 000, whereas the native enzyme M r was found to be 110 000 indicating that the enzyme is a dimer. The enzyme is sensitive to sulfhydryl inhibitions suggesting cysteine residues are essential to enzyme activity. The availability of a homogeneous enzyme preparation should allow further studies on the substrate specificity and the structure and function of the enzyme.

A radioimmunoassay for 6-ketoprostaglandin E 1

A radioimmunoassay for 6-ketoprostaglandin E 1 has been developed. 6-ketoprostaglandin E 1 antibodies were produced in rabbits by repeated immunization with 6-ketoprostaglandin E 1 coupled to bovine serum albumin. [ 125]-labeled hapten with high specific radioactivity was prepared by radioiodination of 6-ketoprostaglandin E 1-tyrosine methyl ester conjugate followed by purification with thin layer chromatography. The antibodies showed good specificity toward 6-ketoprostaglandin E 1 and crossreacted only significantly with prostaglandin E 1. The sensitivity of the assay was 10 pg per assay tube. Application of the radioimmunoassay was demonstrated by the detection of immunoreactive 6-ketoprostaglandin E 1 from 6-ketoprostaglandin E 1α catalyzed by swine renal NADP +-linked 9-hydroxyprostaglandin dehydrogenase.

Radioimmunoassay of Carteolol in Human Plasma

Abstract A radioimmunoassay for the direct measurement of carteolol, a new @-adrenoreceptor blocker, in human plasma was developed. Car- teolol was acylated to form 0-glutarylcarteolol, which was conjugated to bovine serum albumin to provide the immunogen. Antibody to car- teolol was raised in New Zealand albino rabbits. The tracer was the ra- dioiodinated derivative of 0-glutarylcarteolol-tyrosine methyl ester conjugate. The method is highly sensitive, with a lower quantifiable concentration of ∼0.4 ng of carteolol/ml using 0.1 ml of plasma, and has good specificity, with the major metabolite (8-hydroxycarteolol) showing only 0.2% cross-reactivity. It is reproducible, with relative standard de- viations from triplicate standard curves being mostly within ±8%. The method is currently being used to monitor carteolol levels in clinical samples.

Radioimmunological assay of prostacyclin synthetase activity

A radioimmunoassay for 6-oxoprostaglandin F 1α, has been developed. 6-Oxoprostaglandin F 1α antiplasma was produced in rabbits by repeated immunization with 6-oxoprostaglandin F 1α, coupled to bovine serum albumin. [125 I] -labelled hapten with high specific radioactivity was prepared by radioiodination of 6-oxoprostaglandin F 1α-tyrosine methyl ester conjugate followed by purification with thin layer chromatography. The antibodies showed good specificity toward 6-oxoprostaglandin F 1α and crossreacted only significantly with prostaglandin F 1α. The radioimmunoassay was applied to the determination of prostacyclin synthetase activity in swine aorta microsomes using arachidonic acid and prostaglandin H 2 as indirect and direct substrates. The enzyme assay was validated by the criteria that the formation of 6-oxoprostaglandin Fl α, immunoreactivity from either substrates was both time and enzyme protein dependent, and was inhibitable by specific inhibitors of the enzyme system.

Development of radioimmunoassay for thromboxane B 2

A sensitive and specific radioimmunoassay for thromboxane B 2 has been developed. Thromboxane B 2 antiplasma was produced in rabbits by repeated immunization with thromboxane B 2 coupled to bovine serum albumin. [ 125I]-labeled antigen with high specific activity was prepared by radioiodination of thromboxane B 2-tyrosine methyl ester conjugate followed by purification with thin-layer chromatography. Thromboxane B 2 antiplasma cross-reacted less than 0.05% with PGD 2 or PGF 20 and less than 0.01% with other prostaglandins, arachidonic acid, or endoperoxide analogs. The sensitivity of the assay was 5 pg per assay tube.

Thin-layer chromatography of steroid derivatives for radioimmunoassay

Separation by thin-layer chromatography of the intermediates and final products in the synthesis of steroid-protein and steroid-tyrosine methyl ester conjugates is described for the following steroids: aldosterone, cholesterol, cortisol, progesterone and testosterone.