Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Definitive Solution Structures for the 6-Formylated Versions of 1-(βD-Ribofuranosyl)-, 1-(2′-Deoxy-β-D-Ribofuranosyl)-, and 1-β-D-Arabinofuranosyluracil,and of Thymidine

Abstract

ROESY and NOESY NMR spectroscopic analyses of the ribofuranosyl (1a), 2′-deoxyribofuranosyl (1b), and arabinofuranosyl (1c) derivatives of 6-formyluracil in (CD₃)₂SO and D₂O solutions have established that each exclusive 7,05′-cyclic hemiacetal diastereomer of 1a,b and the major 7,O2′-cyclic hemiacetal diastereomer of 1c possess the 7R configuration. In addition, (7R)-1c has been shown to be thermodynamically more stable than (7S)-1c, contrary to our previous indication. A new, higher yielding synthetic route to 1a has been developed, 1b has been obtained for the first time in crystalline form, the route to 1c has been modified to better accommodate large scale preparations, and a new, fourth member of this class, 6-formylthymidine (1d), has been synthesized and its solution structures in (CD₃)₂SO, D₂O, and CD₃OD have been determined. Antitumor and antiviral evaluations of 1a-c have revealed no significant levels of activity.     

Adenosine Receptor Agonists. X-Ray Crystal Structure of Neca 1-(6-Amino-9H-Purin-9-Yl)-1-Deoxy-N-Ethyl-β-D-Ribofuranuronamide

Abstract

The single crystal x-ray structure of NECA (1-(6-amino-9H-pu-rin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide), a 5′-modified adenosine analogue, is reported. Crystallized from methanol as the monohydrate, NECA exists in a syn conformation (sugar syn to purine) in the solid state, consistent with both solution measurements and theoretical calculations. The biological profile of NECA may result from this conformational preference.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

Stereoselective Synthesis of 6-Methyl-9-(2-Deoxy-β-D-Erythro-Pentofuranosyl)purine

Abstract

The coupling of the sodium salt of 6-methylpurine with 2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride in acetonitrile gives the di -O-p-toluoyl protected 9-β nucleoside regio- and stereo-selectively in good yield. Methoxide deprotection followed by preparative hplc then affords pure 6-methyl-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine.     

Preparation of 9-β-D-Arabinofuranosylguanine (araG)

Abstract

A convenient practical synthesis of araG is described.     

Chemical analysis of an immunostimulating (1→4)-, (1→6)-branched glucan from an edible mushroom, Calocybe indica

An immunostimulating water-soluble glucan was isolated from hot aqueous extract of fruit bodies of an edible mushroom Calocybe indica. Structural investigation of the glucan was carried out using acid hydrolysis, methylation analysis, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments, the structure of the repeating unit of the polysaccharide was established as [see figure in text]. This glucan stimulated the splenocytes and thymocytes.     

Synthesis of the Selenium Analog of the Cytokinin 6-(3-Methyl-2-Butenylamino)-2-Methylthio-9-(β-D-Ribofuranosyl)Purine

Abstract

6-(3-methyl-2-butenylamino)-2-methylthio-9-(β-D-ribofuranosyl)purine (1) is a naturally occurring nucleoside with potent cytokinin activity. It has been isolated and identified in the t-RNA of E. coli,^1,2 the t-RNA from wheat germ^3,4 and from Staphylococcus epidermidis.⁵ In addition, compound 1 has been found in t-RNA species corresponding to each of six amino acids whose codons start with uridine, i.e., t-RNA^Cys     

The Inhibition of Adenosine Kinase by α,ω-Di (Adenosin - N 6- YL) Alkanes1

Abstract

Members of a series of α,ω-di(adenosin- N ⁶-yl)alkanes, comprising two adenosine residues linked with alkyl bridges from 1 to 14 methylene units in length, were found to be inhibitors of rat liver and BHK cell adenosine kinase. The inhibition was competitive with respect to adenosine and non-competitive with respect to ATP. The corresponding α,ω-di(cytidin-N ⁴-yl) alkanes were not inhibitors and N ⁶-alkyladenosines inhibited only weakly.     

Synthetic Studies on Nephritogenic Glycosides. Synthesis of β-Glc-(1→6)-α-Glc-(l→6)-α-Glc-(1→Asn)

Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.     

Conformational Studies of Dextran [α-(1⇒6)-D-Glucan]

Abstract

A theoretical conformational study of dextran, a (l?6)-linked α-D-glucan polysaccharide, has been made to allow an explicit comparison with earlier results on pustulan, the corresponding (1 ?6)-linked β-D-glucan. The nonbonded, torsional and hydrogen bond contributions to potential energy were calculated as a function of rotational angles φ, ψ, and ω The (φ, ψ, ω)-space of the disaccharide and of helices contain many local energy minima with very small energy differences. A comparison of (1?6)-α-D-glucans with (1?6)-β-D-glucans indicates significant differences in conformational behavior. Specifically, our results shed light on the fact that dextran does not gel, whereas pustulan does. The difference in tendency to gel may be related to the fact that dextran has no particularly favored conformations with structural regularity whereas pustulan does.     

Superoxide scavenging activity of plastoquinone derivative 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1)

The plastoquinone derivative 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) has the ability to scavenge superoxide anion radical. This ability is manifested both in vitro and in vivo in experiments with the bacterium Escherichia coli. The protective effect of SkQ1 in vivo significantly exceeds that of ascorbic acid.     

AIPL1, a Protein Associated with Childhood Blindness, Interacts with ��-Subunit of Rod Phosphodiesterase (PDE6) and Is Essential for Its Proper Assembly

Mutations in the gene coding for AIPL1 cause Leber congenital amaurosis (LCA), a severe form of childhood blindness. The severity in disease is reflected in the complete loss of vision and rapid photoreceptor degeneration in the retinas of mice deficient in AIPL1. Our previous observations suggest that rod photoreceptor degeneration in retinas lacking AIPL1 is due to the massive reduction in levels of rod cGMP phosphodiesterase (PDE6) subunits (α, β, and γ). To date, the crucial link between AIPL1 and the stability of PDE6 subunits is not known. In this study using ex vivo pulse label analysis, we demonstrate that AIPL1 is not involved in the synthesis of PDE6 subunits. However, ex vivo pulse-chase analysis clearly shows that in the absence of AIPL1, rod PDE6 subunits are rapidly degraded by proteasomes. We further demonstrate that this rapid degradation of PDE6 is due to the essential role of AIPL1 in the proper assembly of synthesized individual PDE6 subunits. In addition, using a novel monoclonal antibody generated against AIPL1, we show that the catalytic subunit (α) of PDE6 associates with AIPL1 in retinal extracts. Our studies establish that AIPL1 interacts with the catalytic subunit (α) of PDE6 and is needed for the proper assembly of functional rod PDE6 subunits.     

An Easy Synthesis of (E)-9-Oxo-2-decenoic Acid,the Queen Substance of the Honey Bee,and (Z)-6-Heneicosen-ll-one,the Sex Pheromone of the Douglas Fir Tussock Moth†

We investigated the effect of dietary phospholipid (PL) concentrate from bovine milk on the epidermis. Thirteen-week-old hairless male and female mice (Hos:HR-1) were separated into two experimental groups, each fed two experimental diets: the control group and the PL group. The mice were given the experimental diets for 6 weeks. Stratum corneum hydration and transepidermal water loss (TEWL) were measured using Corneometer CM825 and Tewameter TM300 (Courage and Khazaka Electronics, Cologne, Germany) at 3 weeks and 6 weeks. After the feeding period, ceramides in stratum corneum were analyzed. We found that stratum corneum hydration and ceramides in the PL group were significantly higher than those in the control group and that TEWL in the PL group tended to decrease.

These results indicate that dietary PL concentrate improves epidermal function by increasing the amount of ceramides, resulting in higher hydration.     

Synthesis,characterization, and biological activity of Nα-(N-fluoresceinthiocarbamoyl)-(Asp1, Ile5)-angiotensin II

A fluorescent analog of angiotensin II was synthesized by reacting fluorescein 5′-isothiocyanate with (Asp¹, Ile⁵)-angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II was purified by chromatography on DEAE-cellulose and Sephadex G-25. Analysis of the analog by thin-layer chromatography, thin-layer electrophoresis, and reversed-phase high-performance liquid chromatography indicated that the analog was free of angiotensin II and fluorescein 5′-isothiocyanate. N-Terminal sequence analysis demonstrated that fluorescein 5′-isothiocyanate reacted with the N-terminal aspartic acid residue of angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II has an absorption maximum at 492 nm, and the value of the molar extinction coefficient, ?, is 7.7 × 10⁴m^?1 cm^?1. The fluorescence emission maximum occurs at 520 nm. Infusion of the analog (0.69 μg/min/kg body wt) directly into the renal artery of an anesthetized rat reduced the blood flow by 12 to 27% within 2 min. Infusion of angiotensin II (0.48 μg/min/kg body wt) reduced renal arterial blood flow by 35 to 53% within 2 min. Saralasin, a partial agonist and antagonist of angiotensin II, inhibited the biologic effect of the fluorescent analog and angiotensin II by 75 and 70%, respectively. The purity, spectral properties, and in vivo biologic activity of N^α-(N-fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II indicate that this analog should facilitate characterization of angiotensin II receptors.     

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.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

Synthesis of 9-β-d-Glucopyranosyl-Adenine-6′-Phosphate

Synthesis of 9-β-d-glucopyranosyl-adenine-6′-phosphate is described. The method developed here involves the process of condensation of base (chloromercuri-6-benzamidopurine) (I) with phosphorylated sugar (2,3,4-tri-O-acetyl-6-diphenylphosphoryl-α-d-glucopyranosyl bromide) (II). This reaction gives crystalline 6-benzamido-9-(2′,3′,4′-tri-O-acetyl-6′-diphenylphosphoryl-β-d-glucopyranosyl)-purine (III) in high yield, which is converted to the desired nucleotide by alkaline hydrolysis.