Nδ-benzoyl-l-ornithine and Nδ-benzoyl-l-γ-hydroxyornithine from Vicia pseudo-orobus

Two new non-protein amino acids, N^δ-benzoyl-l-ornithine and N^δ-benzoyl-l-γ-hydroxyornithine have been characterized from the seeds of Vicia pseudo-orobus.     

A route from d-galactose to the aggregation pheromone component (-)-α-multistriatin

Oxidation of 3,6-di-O-benzyl-1,2-O-isopropylidene-α-d-glucofuranose with pyridinium chlorochromate in the presence of molecular sieves, followed by conversion into the p-tolylsulfonylhydrazone, addition of methyl phenylphosphinate, and reduction with sodium borohydride, provided the key intermediate, namely, 5(R,S)-3,6-di-O-benzyl-5-deoxy-1,2-O-isopropylidene-5-C-[(methoxy)phenylphosphinyl]-α-d-xylo-hexofuranose, in 23% overall yield. Treatment of this compound with sodium dihydrobis(2-methoxyethoxy)aluminate, followed by the action of mineral acid and acetic anhydride, yielded the crystalline title compound, the structure of which was established on the basis of mass and 400-MHz, ¹H-n.m.r. spectra. A general dependence of ²J_PH values on the OPCH dihedral angles effectively served for assigning the configuration of C-1, C-5, and the ring-phosphorus atom of the present product and other such 5-C-phosphinylhexopyranoses.     

Synthesis of 4′-(Hydroxymethyl)Guanosine and a Phosphonate Analogue of Guanylic Acid

Abstract

The synthesis of 4′-(hydroxymethyl)guanosine (7) and the phosphonate analogue 8 of guanylic acid proceed from a common intermediate, 2′, 3′-O-isopropylidene-N ²-(monomethoxytrityl)-guanosine-5′-aldehyde (13).     

α-galactosidase from germinating guar (Cyamopsis tetragonolobus) seeds

The changes in α-galactosidase activity in guar (Cyamopsis tetragonolobus) seeds was followed during seven days of germination. The enzyme activity was maximal on the first day of germination and gradually decreased during subsequent days. On the second day of germination the partially purified enzyme upon ion-exchange chromatography on CM-Sephadex C-50 was resolved into α-galactosidase-A (anionic), α-galactosidase-C₁ (cationic) and α-galactosidase-C₂ (cationic) and their relative proportions were 28,12 and 60%, respectively. The combined α-galactosidase C₁ and C₂ activities increased in the first two days of germination followed by significant decrease after the 3rd day onwards, whereas α-galactosidase-A remained fairly constant throughout the germination period, α-Galactosidase-A and C2 had differentK _m and V_max values withp-nitrophenyl α-D-galactopyranoside, raffinose and melibiose as substrates and also differed in their thermal stabilities     

Stereocontrolled preparation of C-2-deoxy-α- and -β-d-glucopyranosyl compounds from tributyl-(2-deoxy-α- and -β-d-glucopyranosyl)stannanes

tributylstannyllithium treatment of 3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyl chloride (2) provided selectively tributyl (3,4,6-tri-O-benzyl-2-deoxy-β-d-arabino-hexopyranosyl)stannane (3) in 85% yield. Isomeric tributyl (3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyl)stannane (6) could be prepared in 70% yield by reductive lithiation of 2 and reaction with tributyltin chloride. Tin—lithium exchange reaction, performed on 3 and 6 with butyllithium in oxolane at −78°, generated the corresponding, configurationally stable 2-deoxy-β- and -α-d-hexopyranosyllithium compounds which reacted with electrophilic compounds with retention of configuration. Addition to these glycosyllithium reagents to prochiral carbonyl compounds gave variable degrees of facial selectivity. A significant diastereofacial discrimination (10:1) was observed by condensation of 3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyllithium reagent with hexanal and isobutyraldehyde. The structure of all C-glycopyranosyl compounds obtained was established by ¹H-n.m.r. spectroscopy.     

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A_1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K_m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d-glucose into the growing α-d-glucan and the synthesis of high molecular-weight polymers. However, the α-d-glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than IIa.     

α-crystallin-type heat-shock protein from mycoplasma Acholeplasma laidlawii (Mollicutes)

An increased amount of major heat-shock proteins (HSPs) after heat treatment has been revealed in Acholeplasma laidlawii cells grown in liquid culture, with the quantity of small HSPs, known as P17, being enhanced by hundreds times. The P17 protein was isolated and identified as an α-crystallin-type HSP (α-HSP) by sequencing the N-terminal 15 amino acids of the P17 polypeptide chain followed by finding the corresponding open reading frame (ORF) in the completely sequenced genome of A. laidlawii PG 8A. A computer-based search for homologous ORFs in the genomes of all 14 species of the Mycoplasmataceae family (mycoplasmas themselves) that have been completely sequenced to date yields a negative result. However, among the representatives of the Mollicutes (mycoplasma) class, genes encoding α-HSPs were found in two phytoplasma species (Phytoplasmataceae family) and the acholeplasma examined (Acholeplasmataceae family). It is supposed that the presence or absence of α-HSPs in microorganisms might be related to their inhabitancy; representatives of Acholeplasmataceae and Phytoplasmataceae families mostly reside in plant tissues, which is in contrast to the majority of the Mycoplasmataceae family, which lives in animal and human tissues, i.e., use ecological niches with relatively constant temperature.     

Enantiospecific synthesis of (R-(+)-α-lipoic acid from d-glucose

The first enantiospecific synthesis of natural (R)-(+)-α-lipoic acid in 13 steps starting from d-glucose is described.     

Properties of α-galactosidase II2 from Vicia faba seeds

-Galactosidase II² (MW 43 390) from resting Vicia faba L. seeds had been shown to possess d-glucose/d-mannose-specific lectin activity. Inhibition studies with monosaccharides and an examination of the effects of heat and pH on the catalytic and lectin activities of the enzyme indicate that the enzyme substrate and the lectin haptens bind at different sites on the protein. d-Mannosebinding has been investigated by equilibrium dialysis and spectrophotometrically. Both methods yield K_a values of approx. 3·10³ M^-1 for the interaction and there would appear to be two mannosebinding sites per molecule of enzyme protein. The lectin properties of V. faba -galactosidase II² have been discussed in relation to both V. faba lectin (favin) and other legume -galactosidases.Abbreviations con A concanavalin A - CM-cellulose carboxymethyl cellulose - MW molecular weight - PNPG p-nitrophenyl -d-galactoside - SDS sodium dodecyl sulphate - PAGE polyacrylamide-gel electrophoresis     

Purification and properties of α-galactosidases from Vicia faba seeds

Two forms of alpha-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2.8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. alpha-Galactosidases I and II showed different pH optima and K(m) and V(max.) values with p-nitrophenyl alpha-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.     

Antitumor properties of substituted (αE)-α-(1H-indol-3-ylmethylene)benzeneacetic acids or amides

A novel class of indole derivatives characterized by a (αE)-α-(1H-indol-3-ylmethylene)benzeneacetic acid or amide scaffold was synthesized. These derivatives, assayed for cell-growth inhibition activity against a panel of six different tumor cell lines, showed strong antiproliferative activity and selectivity mainly towards DU145 cell line. In particular, compounds 2d–m and 5 stand out for their cell growth inhibitory activity and, among them, compound 2d emerged for its selectivity towards DU145 with respect to other tested tumor cell lines. DU145 treated with 1 μM of 2d for 72 h showed p21^Cip1 induction and suppression of Akt signaling together with induction of Rb. From a computational point of view, two different approaches were used in order to study topology and electronic properties of the novel compounds and to shed light on their drug-likeness properties. Firstly, topological and electronic features of the compounds endowed with the most relevant biological activity were deepened; in parallel, some ADME properties like solubility and permeability were predicted.     

α-D-galactose-specific lectin from jack fruit (A rtocarpus integra) seed

An α-D-galactose-specific lectin from the seeds of jack fruit (Artocarpus integra) has been isolated in pure form by affinity chromatography on immobilised guar gum (a galactomannan). The lectin is shown to be a glycoprotein containing 3% carbohydrate and having a molecular weight of 39,500 as determined by gel filtration. Sodium dodecyl sulphate gel electrophoresis revealed a single polypeptide of 10,500 dalton, indicating that the native lectin is a tetrarner of identical subunits. The hemagglutinating activity of the lectin towards erythrocytes of all blood groups is found to be the same.     

Production of β-(pyrazol-1-yl)-l-alanine from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A

-(Pyrazol-1-yl)-l-alanine (-PA) was produced from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A. The amount of -PA increased with increasing l-serine concentrations up to 600 mM at 50 mM pyrazol while 100 mM pyrazol gave the highest -PA production with 50 mM l-serine. Under the optimized conditions, -PA accumulated in the broth at approximately 140 mM with a conversion of 90% with respect to the added amount of pyrazol.     

Macluralisin — a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid.

A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.Abbreviations DFP diisopropylfluorophosphate - PMSF phenylmethylsulfonylfluoride - Glp pyroglutamyl - NHC₆H₄NO₂ p-nitroanilide This work was supported in part by a grant from the Russian Foundation for Basic Research.     

蚕豆(Vicia faba)M染色体DNA复制顺序的研究

真核细胞中,染色体DNA由许多复制子组成。一个复制子所需的复制时间短于S期时长,这意味着染色体DNA的各个复制子并非同时复制,而是按某种先后次序进行复制的。许多学者以人和动物细胞为材料进行了有关染色体DNA复制顺序的研究,但是关于植物染色体DNA复制顺序的研究报道还不很多。植物根端分生组织细胞在周期时长方面差异很大。Evans曾经研究过蚕豆根端细胞染     

Biotransformation of (-)-(R)-α-Phellandrene: Antimicrobial Activity of Its Major Metabolite

Terpene derivatives converted by microbial biotransformation constitute an important resource for natural pharmaceutical, fragrance, and aroma substances. In the present study, the monoterpene α-phellandrene was biotransformed by 16 different strains of microorganisms (bacteria, fungi, and yeasts). The transformation metabolites were initially screened by TLC and GC/MS, and then further characterized by NMR spectroscopic techniques. Among the six metabolites characterized, 6-hydroxypiperitone, α-phellandrene epoxide, cis-p-menth-2-en-1-ol, and carvotanacetone, which originated from (-)-(R)-α-phellandrene, are reported for the first time in this study. Additionally, the substrate and the metabolite 5-p-menthene-1,2-diol were subjected to in vitro antibacterial and anticandidal tests. The metabolite showed moderate-to-good inhibitory activities (MICs=0.125 to >4?mg/ml) against various bacteria and especially against Candida species in comparison with its substrate (-)-(R)-α-phellandrene and standard antimicrobial agents.     

Synthesis of p-trifluoroacetamidophenyl O-α-D-galactopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→4)-α-L-rhamnopyranoside

The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N-acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δk_?1/k₊₁), and kinetic parameters (K_m,k₂) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10^?1min^?1. Acetylation increased the apparent K_m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k² remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.