Asymmetric Animation I. Asymmetric Synthesis of Aspartic Acid and Evaluation of Optical Yield in the Addition of Chiral Phenethylamine to α,β-Unsaturated Esters

The enantioselective addition of chiral benzylic amines to dimethyl maleate and fumarate gives optically active aspartic acid in satisfactory yields. The re-face favorably meets the stereochemical requirements of the chiral R-amines than does the si-face. The stereoselectivity in the present reaction is little dependent on solvent polarity but is significantly enhanced by low temperature. The direct determination by gas-liquid chromatography of the diastereomeric addition products provides more precise values of optical yield and is preferred to the polarimetry of the end product after many steps of transformation with probable fractionations.     

Fungal Reduction of C6 α,β-Unsaturated Carboxylic Acids

The metabolism of sorbic acid (trans-2,trans-4-hexadienoic acid) and its related compounds by Mucor sp. A-73 was investigated. Sorbic acid was reduced by this fungus to trans-4-hexenol (more than 90% yield). In a series of hexamonoenoic acids, carboxyl groups and α,β-double bond were reduced, but β,γand γ,δ double bonds were hardly reduced. The reduction of cis-2-hexenoic acid was slower than that of the corresponding trans isomer. Sorbic alcohol, one of α,β-unsaturated alcohols, was converted well to trans-4-hexenol by the fungus. These results showed that this fungus could carry out two independent reductions: (i) carboxyl group→alcohol, (ii) α,β-unsaturated alcohol→αβ-saturated one. Furthermore, α,β-unsaturated alcohols were temporarily detected in the course of fungal reductions of some α,β-unsaturated acids. The fact suggested that the reduction of α,β-unsaturated acids to α,β-saturated alcohols was initiated by the reaction (i) and followed by (ii). The biological hydrogenation of α,β-unsaturated alcohols is a new reaction.     

Synthesis of 3′-C-Phosphono Nucleosides from α,β-Unsaturated Lactone

Abstract

The reaction of 5-protected α,β-unsaturated γ-lactone 4 with trialkylphosphite gave 3′-C-dialkylphosphono-erythro lactone 5 in high yields. The lactone 5 was reduced with DIBAL to the corresponding lactol, which was converted to the acetate 6 by treatment with acetic anhydride in pyridine. The acetate 6 was coupled with silylated thymine in the presence of TMS-triflate and the resulting anomeric mixture of nucleotides could be separated chromatographically and after desilylation using TBAF in THF the 3′-C-dialkylphosphono nucleosides 7 and 8 were obtained.     

A New Class of Pyrethroidal Insecticides; α-Substituted Phenylacetic Acid Esters

A new nucleoside antibiotic, mildiomycin D, was isolated from the culture broth of Streptoverticillium rimofaciens B-98891 as a minor component. The molecular formula of the antibiotic purified by silica gel and ion exchange resin column chromatographies was determined to be C₁₉H₃₀N₈O₈ ? (2H₂O) from its physicochemical data. The ultraviolet and infrared spectra were very similar to those of mildiomycin, a major component. On the basis of ¹H and ¹³C-NMR spectra and acidic hydrolysates of the compound, the chemical structure of the antibiotic was determined as a deoxy compound at the C_8′ position in mildiomycin. Mildiomycin D showed weak activities against Gram-positive and negative bacteria, phytopathogenic fungi and some yeasts, and its activity against Rhodotorula rubura was about 40% that of mildiomycin.     

The Application of α-Picolinic Acid on the Screening of Rice Blast-Resistant Variants

The screening of rice (Oryza sativa L.) variants resistant to blast was studied by using α-picolinic acid treatment. In order to establish the selection conditions, anthers and anther-derived calla of rice strain C2, which is susceptible to blast, were cultured ma media supplemented with different concentrations of α-picolinic acid to examine the response to α-picolinic acid. it was shown that callus induction frequency of anther and callus growth were obviously declined in relevance to the increasing the concentrations of α-picolinic acid. 40 mg/L of α-picolinic acid was the crucial concentration for anther-derived callus induction and growth of the rice strain C2. The callus lines tolerated to 40 mg/L of α-picolinic acid were selected from rice strain C2 and then regenerated into green plants. By identification of blastresistance in the seedling and boot stage of R1 and R2 generations, two variants No. 4-091 and No. 2-07 resistant to Pyricularia oryzae strain ZA1 and ZB11 were obtained, however, the grain yield per plant of these variants were not so high as C2.     

Effect of Rifampin on the Development of Ribonucleic Acid Bacteriophage Qβ

The drug rifampin, when added at the time of infection, inhibits synthesis of the phage Qbeta. Both viral ribonucleic acids and viral proteins are made in nearly the same amount as in the absence of rifampin, but the rate of assembly into phage particles is low.     

Synthesis and Anticancer Activity of Novel Derivatives of α,β-Unsaturated Ketones Based on Oleanolic Acid: in Vitro and in Silico Studies against Prostate Cancer Cells

Herein, new derivatives of α,β-unsaturated ketones based on oleanolic acid ( 4 a – i ) were designed, synthesized, characterized, and tested against human prostate cancer (PC3). According to the in vitro cytotoxic study, title compounds ( 4 a – i ) showed significantly lower toxicity toward healthy cells (HUVEC) in comparison with the reference drug doxorubicin. The compounds with the lowest IC₅₀ values on PC3 cell lines were 4 b (7.785 μM), 4 c (8.869 μM), and 4 e (8.765 μM). The results of the ADME calculations showed that the drug-likeness parameters were within the defined ranges according to Lipinski's and Jorgensen's rules. For the most potent compounds 4 b , 4 c , and 4 e , a molecular docking analysis using the induced fit docking (IFD) protocol was performed against three protein targets (PARP, PI3K, and mTOR). Based on the IFD scores, compound 4 b had the highest calculated affinity for PARP1, while compound 4 c had higher affinities for mTOR and PI3K. The MM-GBSA calculations showed that the most potent compounds had high binding affinities and formed stable complexes with the protein targets. Finally, a 50 ns molecular dynamics simulation was performed to study the behavior of protein target complexes under in silico physiological conditions.     

Microbial Degradation of α-Picolinic Acid, 2-Pyridinecarboxylic Acid

The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30% raw winged bean diet caused strong growth depression in young rats, and led to death within 10 ~ 20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% l-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.     

Effects of Suramin on the ATP- and α,β-Methylene-ATP-induced Constriction of the Rabbit Ear Artery

Abstract

The isolated rabbit ear artery contains both dilation-mediating P_2Y-receptors and constriction-mediating P_2X-receptors. Suramin antagonizes the effects of ATP at either receptor.     

The Influence of Cosolvent on the Complexation of HP-β-cyclodextrins with Oleanolic Acid and Ursolic Acid

The present work was aimed at the influence of ethanol on the complex formation of hydroxypropyl-β-cyclodextrin (HP-β-CD) with oleanolic acid (OA) and ursolic acid (UA), two insoluble isomeric triterpenic acids. Phase solubility studies were carried out to evaluate the solubilizing power of HP-β-CD, in association with ethanol, toward OA and UA. A mathematical model was applied to explain and predict the solubility of OA and UA influenced by HP-β-CD and ethanol. The solid complexes were prepared by evaporating the filtrate of samples which was prepared in different complexing media. The solubility of OA is much higher than that of UA in all the tested aqueous solutions. The solubility of OA and UA can be increased over 900 and 200 times, respectively, by forming complex with HP-β-CD. Ethanol (0.5%, v/v) can help the formation of OA-HP-β-CD complex, but is harmful to the formation of UA-HP-β-CD complex. Increasing solubility in water can be achieved by adding ethanol into the complexing media, but the concentration of ethanol should be optimized. The ring E of the chemical compounds has a great influence on the complexing process.     

Influence of pH on the Structure and Oleic Acid Binding Ability of Bovine α-Lactalbumin

The biological function of ??-lactalbumin (??-LA) depends on its conformation. ??-LA can adopt a stable intermediate state induced by heating or pH change. This intermediate state associates with oleic acid (OA) to form an anti-tumor complex. The effect of temperature on the formation the complex has been studied, whereas the effect of pH on complex formation remains unresolved. The effect of pH on tryptophan residues, hydrophobic clusters and secondary structure of Ca²⁺-depleted bovine ??-LA (BLA) was studied by fluorescence spectroscopy and circular dichroism. BLA was found to adopt a more flexible conformation between pH 7.0 and 9.0 with buried hydrophobic clusters. The binding ability of ??-LA towards OA and the anti-tumor activity of the corresponding complex were also studied. BLA was found to bind more OA over the pH range of 7.0?C9.0 and the corresponding complexes showed a higher anti-tumor activity than those complexes formed under acidic conditions. Our study indicates that alkaline pH aided the formation of the complex as well as its anti-tumor activity. We also propose a possible characteristic structure that facilitates binding of OA.     

Inhibition of D-amino Acid Oxidase by α-Keto-γ-Methiolbutyric Acid,product of the reaction with D-Methionine

Summary The inhibition of D-Amino Acid Oxidase by -Keto Acid, product of the reaction with D-Amino Acid is described for the first time. Inhibition of the enzyme by -Keto--Methiolbutyric Acid (4-methylthio-2-oxobutanoic acid), product of the reaction with D-Methionine, was studied. From the results obtained it is deduced that inhibition is competitive with the substrate, the value of the inhibition constant being 1.85 10^–3 M.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

The Synthesis of Dimethyl Esters of dl-O,O′-Dimethylfukiic Acid and dl-O,O′-Dimethylepifukiic Acid

The synthesis of dimethyl esters of dl-O,O′-dimethylfukiic acid (11) and dl-O,O′-dimethylepifukiic acid (12) are described.     

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization.     

Oligomerization of α-Thioglutamic Acid

A decaglutamic acid primer is extended very slowly by-thioglutamic acid. The addition of bicarbonate to thereaction mixture greatly accelerates the reaction. We believethe N-carboxyanhydride of glutamic acid is an intermediate inthe accelerated reaction. When K₃Fe(CN)₆ andbicarbonate are both present in the reaction mixture,oligoglutamic acids up to at least the 15-mer are formedrapidly. The acylating agent is the oxidation product ofthioglutamic acid, a diacyldisulfide.     

Red Pigment Produced by the Reaction of Dehydro-l-ascorbic Acid with α-Amino Acid

At the initial stage of the browning reaction of dehydro-l-ascorbic acid (DHA) with α-amino acid, a kind of red pigment was produced. The pigment was isolated as very hygroscopic red powder from non-aqueous reaction system, and its characterization was made. It was revealed that it had the same structure with that of the red pigment produced by the oxidation of l-scorbamic acid, an intermediate amino-reductone expected to be produced by Strecker degradation. Formation mechanism of the pigment which was considered to be an intermediate of browning reaction of DHA with α-amino acid was also discussed.     

Stimulatory effect of α-synuclein on the tau-phosphorylation by GSK-3β

Hyperphosphorylation of tau protein (tau) causes neurodegenerative diseases such as Alzheimer's disease (AD). Recent studies of the physiological correlation between tau and α-synuclein (α-SN) have demonstrated that: (a) phosphorylated tau is also present in Lewy bodies, which are cytoplasmic inclusions formed by abnormal aggregation of α-SN; and (b) the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) increases the phosphorylation of tau as well as the protein level of α-SN in cultured neuronal cells, and also in mice. However, the molecular mechanism responsible for the α-SN-mediated hyperphosphorylation of tau remains to be elucidated. In this in vitro study, we found that: (a) α-SN directly stimulates the phosphorylation of tau by glycogen synthase kinase-3β (GSK-3β), (b) α-SN forms a heterotrimeric complex with tau and GSK-3β, and (c) the nonamyloid beta component (NAC) domain and an acidic region of α-SN are responsible for the stimulation of GSK-3β-mediated tau phosphorylation. Thus, it is concluded that α-SN functions as a connecting mediator for tau and GSK-3β, resulting in GSK-3β-mediated tau phosphorylation. Because the expression of α-SN is promoted by oxidative stress, the accumulation of α-SN induced by such stress may directly induce the hyperphosphorylation of tau by GSK-3β. Furthermore, we found that heat shock protein 70 (Hsp70) suppresses the α-SN-induced phosphorylation of tau by GSK-3β through its direct binding to α-SN, suggesting that Hsp70 acts as a physiological suppressor of α-SN-mediated tau hyperphosphorylation. These results suggest that the cellular level of Hsp70 may be a novel therapeutic target to counteract α-SN-mediated tau phosphorylation in the initial stage of neurodegenerative disease.     

Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.