Isolation and analysis by the reductive-cleavage method of linkage positions and ring forms in the Mycobacterium smegmatis cell-wall arabinogalactan

The Mycobacterium smegmatis arabinogalactan polysaccharide has been isolated from the cell wall by saponification and extraction to remove lipids and subsequent solubilization by treatment with lysozyme. Analysis for neutral sugars demonstrated the presence of D-arabinose and D-galactose in a ratio of 3:1, respectively. Reductive cleavage of the fully methylated polysaccharide in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate and subsequent acetylation in situ gave six partially methylated 1,4-anhydroalditol acetates as the major products and three partially methylated 1,5-anhydroalditol acetates as minor products. Partially methylated 1,5-anhydroalditol acetates were not formed when reductive cleavage was accomplished with triethylsilane and a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate as the catalyst, demonstrating that the polysaccharide is exclusively comprised of furanosyl residues. The partially methylated anhydroalditols so produced were identified by comparison to authentic standards. Their identifies are consistent with the presence in the M. smegmatis arabinogalactan of an octasaccharide repeating unit comprised of a nonreducing terminal D-arabinofuranosyl group, a 2-O-linked D-arabinofuranosyl residue, three 5-O-linked D-arabinofuranosyl residues, a 3,5-di-O-linked D-arabinofuranosyl residue, a 5-O-linked D-galactofuranosyl residue, and a 6-O-linked D-galactofuranosyl residue.     

Bidirectional transformation of aromatic aldehydes by Desulfovibrio desulfuricans under nitrate-dissimilating conditions

M. PAREKH, H.L. DRAKE AND S.L. DANIEL 1996. Desulfovibrio desulfuricans ATCC 27774 was screened for reactivity against aromatic compounds during lactate-dependent, nitrate-dissimilating growth. Only aromatic aldehydes (benzaldehyde, 2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, vanillin, iso -vanillin and o -vanillin) were reactive and, with the exception of 2-hydroxybenzaldehyde, were stimulatory to lactate-dependent growth. Aromatic aldehydes were transformed to their corresponding benzoate and benzyl alcohol derivatives, with the ratio of benzoate-to-benzyl alcohol derivatives being dependent upon lactate availability. In presence of lactate, aromatic aldehydes were primarily reduced to their corresponding benzyl alcohol derivatives; in the absence of lactate, aromatic aldehydes were mainly oxidized to their corresponding benzoate derivatives. In the absence of nitrate, 3-hydroxybenzaldehyde was neither reduced nor oxidized. These results indicate that D. desulfuricans is competent in the bidirectional transformation of aromatic aldehydes under nitrate-dissimilating conditions and that the direction of transformation (i.e. reduction or oxidation) is regulated by reductant availability.     

Design and synthesis of indole and tetrahydroisoquinoline hydantoin derivatives as human chymase inhibitors

The synthesis of new potential inhibitors of human chymase is described. Treatment of dihydroimidazo[1,5-a]indole and [1,5-b]isoquinoline-dione with thioaryl followed by oxidation gave the N-arylsulfonylmethyl of polycyclic hydantoin derivatives 3, 5 and 6.     

Increased urinary excretion of 1-methyl-2-pyridone-5-carboxamide in rats administered 2-acetylaminofluorene.

The onset of fat accumulation within CCl₄ poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated.The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [³H]colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes or LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active.The presence of thiols, added to the incubation medium, partially protects against the inhibition of [³H]colchicine binding by aldehydes. This suggests that aldehydes act by reacting with -SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CCl₄ poisoning is discussed.     

Convenient and Efficient Syntheses of N 6- and N 4- Substituted Adenines and Cytosines and their 2′-Deoxyribosides

Convenient and efficient methods of the synthesis of N⁶- and N⁴-substituted derivatives of adenine and cytosine and their 2′-deoxyribosides were developed. The reactions of either unprotected nucleobases (adenine, cytosine) or unprotected 2′-deoxyribosides with aryl or alkyl aldehydes give corresponding Schiff bases that can be reduced to the target title compounds with high overall yields. In the case of aryl aldehydes the imine derivatives are obtained in the presence of methoxides in methanol and reduced with sodium borohydride. The corresponding reactions with alkyl aldehydes require the use of acetic acid and borane dimethyl sulfide complex instead.     

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6: SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND ANALYSIS OF REACTION STEPS*

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH₃CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (^Dk_app ∼ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The ^Dk_app for DEN was ∼3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO₂H and CH₃CO₂H, respectively. In neither case was a lag observed, which was unexpected considering the k_cat and K_m parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).     

Design and Synthesis of Indole and Tetrahydroisoquinoline Hydantoin Derivatives as Human Chymase Inhibitors

The synthesis of new potential inhibitors of human chymase is described. Treatment of dihydroimidazo[1,5-a]indole and [1,5-b]isoquinoline-dione with thioaryl followed by oxidation gave the N-arylsulfonylmethyl of polycyclic hydantoin derivatives 3, 5 and 6.     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

Synthesis of 1,5-Anhydro-D-Mannitol Nucleosides with a Purine Base Moeety

Abstract

D-Mannitol nucleosides with a purine base moiety have been conveniently synthesized strating from 1,5-anhydro-4,6-O-benzylidene-D-glucitol. The 3-OH function of 1,5-anhydro-4,6-O-benzylidene-D-glucitol was selectively protected with t-butyldimethylsilyl group and subsequently converted to the corresponding 0-triflate derivative for the introduction of the nucleobase moietes. These nucleoside derivatives were transformed to 1,5-Anhydro-6-O-MMTr-2-(N⁶-benzoyladenin-9-yl)-2-deoxy-3-O-TBDMS-D-mannitol and 1,5-Anhydro-6-O-MMTr-2-(N²-isobutyryl-guanin-9-yl)-2-deoxy-3-O-TBDMS-D-mannitol, useful as the building blocks for oligonucleotide synthesis. Also, the synthesis of the corresponding fully deprotected anhydrohexitol nucleosides were achieved for evaluation of antiviral activity test.     

Long-chain bases in the sphingolipids of atherosclerotic human aorta

Long-chain bases were prepared from human aorta sphingomyelin by a combined enzymatic hydrolysis-alkaline hydrolysis procedure and these bases were isolated by thin-layer chromatography. Aldehydes, obtained from the long-chain bases by periodate oxidation, were converted to 1,3-dioxolane derivatives. Dioxolanes were identified and quantified by gas-liquid chromatography before and after catalytic hydrogenation, and before and after separation into saturated, monoene, and diene dioxolane fractions. The monoene dioxolanes were converted to aldehydes by reductive ozonolysis with dimethyl sulfide and these aldehydes were isolated and identified as dioxolane derivatives. The double bond positions in the major diene component were established by reductive ozonolysis and permanganate-periodate oxidation. Sphingenines in the cerebroside-sulfatide and sulfatide fractions of aorta were converted to aldehydes by the reductive ozonolysis of intact sphingolipids and these aldehydes were analyzed as the dioxolanes. Human aorta sphingomyelin contained significant amounts of 4-hexadecasphingenine, 4-heptadecasphingenine, sphinganine, 4-sphingenine, and 4,x14-sphingadienine. Small amounts of hexadecasphinganine, 4-tetradecasphingenine, a sphingadienine isomer, an unknown sphinganine, and two unknown diene long-chain bases were also found in sphingomyelin. The presence of a branched-chain 4-sphingenine was tentatively established and the possible presence of a sphingenine isomer was suggested. The major sphingenines were the same in the sphingomyelin, sulfatide, and cerebroside-sulfatide fractions of human aorta.     

Mouse hepatic microsomal oxidation of aliphatic aldehydes (C8 to C11) to carboxylic acids.

Addition of saturated and alpha, beta-unsaturated aliphatic aldehydes (C8 to C11) significantly increased NADPH oxidation with mouse hepatic microsomes, and the aldehydes themselves were oxidized to the corresponding carboxylic acids. When these aldehyde substrates were incubated similarly under oxygen-18 gas and the carboxylic acids formed were analyzed by GC-MS after methylation, it was indicated that oxygen-18 was significantly incorporated into the carboxylic acids formed from alpha, beta-unsaturated aldehydes, but not significantly into the carboxylic acids formed from saturated aldehydes. These results indicate that enzyme and/or mechanism responsible for the oxidation of these two types of aldehydes is different from each other.     

Bioinorganic and bioorganic studies of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (E.C.1.1.1.1) is an NAD⁺/NADH dependent enzyme with a broad substrate specificity being active on an assortment of primary and secondary alcohols. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones as well as the oxidation of certain aldehydes to their related carboxylic acids. Although the bioinorganic and bioorganic aspects of the enzymatic mechanism, as well as the structures of various ternary complexes, have been extensively studied, the kinetic significance of certain intermediates has not been fully evaluated. Nevertheless, the availability of computer-assisted programs for kinetic simulation and molecular modeling make it possible to describe the biochemical mechanism more completely. Although the true physiological substrates of this zinc metalloenzyme are unknown, alcohol dehydrogenase effectively catalyzes not only the interconversion of all-trans-retinol and all-trans-retinal but also the oxidation of all-trans-retinal to the corresponding retinoic acid. Retinal and related vitamin A derivatives play fundamental roles in many physiological processes, most notably the vision process. Furthermore, retinoic acid is used in dermatology as well as in the prevention and treatment of different types of cancer. The enzyme-NAD⁺-retinol complex has an apparent pK_a value of 7.2 and loses a proton rapidly. Proton inventory modeling suggests that the transition state for the hydride transfer step has a partial negative charge on the oxygen of retinoxide. Spectral evidence for an intermediate such as E-NAD⁺-retinoxide was obtained with enzyme that has cobalt(II) substituted for the active site zinc(II). Biophysical considerations of water in these biological processes coupled with the inverse solvent isotope effect lead to the conclusion that the zinc-bound alkoxide makes a strong hydrogen bond with the hydroxyl group of Ser48 and is thus activated for hydride transfer. Moderate pressure accelerates enzyme action indicative of a negative volume of activation. The data with retinol is discussed in terms of enzyme stability, mechanism, adaptation to extreme conditions, as well as water affinities of substrates and inhibitors. Our data concern all-trans, 9-cis, 11-cis, and 13-cis retinols as well as the corresponding retinals. In all cases the enzyme utilizes an approximately ordered mechanism for retinol–retinal interconversion and for retinal–retinoic acid transformation.     

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.     

Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase

The interaction of trizine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively. Only the substrate activity of 10-(4-chloro-6-methyithio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 × 10^?30 m³. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 × 10^?30 m³ or greater. All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.     

A spectrophotometric assay for monoamine oxidase activity with 2, 4-dinitrophenylhydrazine as a derivatized reagent

A simple, rapid and reliable spectrophotometry was developed to determine monoamine oxidase (MAO). In this study, 2,4-dinitrophenylhydrazine (DNPH), a classic derivatizing reagent, was used to detect MAO-dependent aldehyde production; and traditional DNPH spectrophotometry was simplified. Benzylamine and serotonin oxidation were catalyzed by MAO-B and MAO-A, respectively, to aldehydes. These were derivatized with DNPH, and the corresponding quinones were further formed by adding NaOH. These DNPH derivatives with large conjugated structures were directly measured spectrophotometrically at 465 nm and 425 nm, without the need for precipitating, washing and suspending procedures. The addition of NaOH caused a red shift of the maximum absorption wavelength of these derivatives, which reduced the interference of free DNPH. MAO-B protein was as low as 47.5 μg in rat liver with correlation coefficients ranging within 0.995–0.999. This method is 2–3 times more sensitive than direct spectrophotometry. The detection of MAO inhibition through this method showed that IC₅₀ values of rasagiline are 8.00 × 10⁻⁹ M for MAO-B and 2.59 × 10⁻⁷ M for MAO-A. These results are similar to the values obtained by direct spectrophotometry. Our study suggests that DNPH spectrophotometry is suitable to detect MAO activity, and has the potential for MAO inhibitor screening in the treatment of MAO-mediated diseases.     

A revision of the structure of cucurbitacin S from Bryonia dioica

¹H NMR studies at 400 MHz on cucurbitacin S and its derivatives suggest that the structure of cucurbitacin S previously isolated from Bryonia dioica should be revised from (22S)-22, 25-anhydro-2,16α,22,25-tetrahydroxy-3,11,23-trioxo-cucurbita-1,5-diene to (24S)-16,24-anhydro- 2,16α,24,25-tetrahydroxy-3,11,22-trioxo-cucurbita-1,5-diene.     

Determination of oil oxidation by an aldehyde-requiring mutant of luminous bacteria

A simple, rapid bioluminescence test (BT) for the determination of lipid oxidation is described. The test utilizes an aldehyde-requiring dark mutant of Vibrio harveyi (M42) that emits light in the presence of long chain (C₈-C₁₆) aliphatic aldehydes. The procedure consists of treating the oil or fat with Co²⁺ ion in ethanolic medium at alkaline pH. This treatment facilitates the decomposition of the hydroperoxides into long-chain aldehydes, part of which is used by the bacteria to produce light. The test was evaluated with corn, soybean and safflower oils, and shows excellent correlation with the commonly used peroxide value assay.     

Pathway of n-Alkane Oxidation in Cladosporium resinae

Pathways of initial oxidation of n-alkanes were examined in two strains of Cladosporium resinae. Cells grow on dodecane and hexadecane and their primary alcohol and monoic acid derivatives. The homologous aldehydes do not support growth but are oxidized by intact cells and by cell-free preparations. Hexane and its derivatives support little or no growth, but cell extracts oxidize hexane, hexanol, and hexanal. Alkane oxidation by extracts is stimulated by reduced nicotinamide adenine dinucleotide (phosphate). Alcohol and aldehyde oxidation are stimulated by nicotinamide adenine dinucleotide (phosphate), and reduced coenzymes accumulate in the presence of cyanide or azide. Extracts supplied with (14)C-hexadecane convert it to the alcohol, aldehyde, and acid. Therefore, the major pathway for initial oxidation of n-alkanes is via the primary alcohol, aldehyde, and monoic acid, and the system can act on short-, intermediate-, and long-chain substrates. Thus, filamentous fungi appear to oxidize n-alkanes by pathways similar to those used by bacteria and yeasts.     

An Efficient Route to Novel 4,5‐Di‐ and 2,4,5‐Tri Substituted Imidazoles from Imidazo[1,5‐a]‐1,3,5‐triazine (5,8‐Diaza‐7,9‐dideazapurine) Derivatives

Two new types of imidazole derivatives: N‐(2‐R¹‐5‐R²‐1H‐imidazol‐4‐yl) thioureas 7a–g and N‐(2‐R¹‐5‐R²‐1H‐imidazol‐4‐yl) formamides 8b,c,g were obtained in high yields by the hydrolytic degradation of 6‐R¹‐8‐R²‐2‐thioxo‐2,3‐dihydroimidazo[1,5‐a]‐1,3,5‐triazin‐4(1H)‐ones 5a–g and 6‐R¹‐8‐R²‐imidazo[1,5‐a]‐1,3,5‐triazin‐4(3H)‐ones 6b,c,d, respectively. The tautomeric preferences of the new imidazoles were determined.     

1,5-Anhydro-4-deoxy-d-glycero-hex-1-en-3-ulose and other pyrolysis products of cellulose

Uncatalyzed pyrolysis of cellulose provides a tar containing mainly 1,6-anhydro-d-glucose derivatives and some unsaturated products. The latter include a new enone that has been isolated by preparative column chromatography in 1.4% yield and identified as 1,5-anhydro-4-deoxy-d-glycero-hex-l-en-3-ulose. This compound is also formed by pyrolysis of other carbohydrate polymers. A mechanism for its production from internal units has been deduced from the experimental data. The pyrolysis products of cellulose also contain 3,5-dihydroxy-2-methyl-4H-pyran-4-one, which appears to be an oxidation product.