Rat liver aryl sulfotransferase-catalyzed sulfation and rearrangement of 9-fluorenone oxime

The role of hepatic cytosolic aryl sulfotransferase (3'-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1) in the enzymic rearrangement of 9-fluorenone oxime to phenanthridone was investigated. 9-Fluorenone oxime was found to be an excellent substrate for a partially purified rat liver aryl sulfotransferase preparation. This compound was in fact superior to 2-naphthol, the standard assay substrate. This is the first reported observation of aryl oxime sulfation by the aryl sulfotransferases. 9-Fluorenone oxime sulfation exhibited pronounced substrate inhibition at high substrate concentrations. However, despite virtually complete conversion of 9-fluorenone oxime to the corresponding N-O-sulfate conjugate in enzyme incubation mixtures, only small amounts of rearrangement product were detected after long-term incubations. In addition, 9-fluorenone oxime-O-sulfonic acid was chemically synthesized and tested for stability. The results showed that rearrangement was pH-dependent and occurred slowly over several hours. It is therefore concluded that aryl sulfotransferase-catalyzed sulfation likely plays an important role in the in vitro and in vivo disposition of 9-fluorenone oxime. Moreover, sulfation facilitates the Beckmann-like conversion of 9-fluorenone oxime to phenanthridone. Sulfation alone, however, appears insufficient to account for all of the previously reported in vitro and in vivo rearrangement.     

Synthesis, photophysical, photochemical, DNA cleavage/binding and cytotoxic properties of pyrene oxime ester conjugates

A new series of (E)-pyrene oxime ester conjugates of carboxylic acids including amino acids were synthesized by coupling with an environment sensitive fluorophore 1-acetylpyrene. (E)-Pyrene oxime esters exhibited strong fluorescence properties and interestingly their fluorescence properties were found to be highly sensitive to the surrounding environment. Direct irradiation of the (E)-pyrene oxime esters by UV light (≥350 nm) resulted in both the photo-Beckmann rearrangement product and products resulting from N-O bond homolysis. Photoproduct formation and their distribution were found to be solvent dependent. Further, we also showed (E)-pyrene oxime esters intercalated into DNA efficiently and photo-cleaved DNA. Finally we also showed these oxime esters can permeate cells efficiently and may cause cytotoxicity upon irradiation of light.     

Beckmann reactions of steroidal spirocyclic oximes derived from the 16beta,23:23,26-diepoxy-22-oxo moiety

The Beckmann rearrangement of the syn and anti isomers of the spirocyclic oxime derived from a 16β,23:23,26-diepoxy-5β-cholestan-22-one was studied. Whereas the anti isomer always follows the Beckmann fragmentation course, the syn isomer, depending on the reaction conditions, follows the normal Beckmann rearrangement course and/or the isomerization to the anti isomer followed by the fragmentation course.     

Nitric Oxide Emissions from Soybean Leaves during in Vivo Nitrate Reductase Assays

Recent work identified acetaldehyde oxime as the predominant product purged by inert gases from anaerobic in vivo nitrate reductase (NR) assays of soybean (Glycine max [L.] Merr.) leaves. Another recent study supported earlier research findings which identified the primary product evolved from soybean leaves as nitric oxide (NO). This paper provides evidence that eliminates acetaldehyde oxime and confirms that NO is the primary nitrogenous product purged from the in vivo NR assay system. A portion of the evidence is based on the high water solubility of acetaldehyde oxime. Other evidence presented is the failure by chemical and spectrophotometric means to detect oximes in gases emitted in the purging of the reaction medium or in the leaf tissues. The gaseous product from the in vivo NR assay system reacted identically to NO standards and did not resemble acetaldehyde oxime standards. It was concluded that the predominant N product within the leaves was nitrite and that the predominant gaseous N product evolved from the assay was NO.     

Steroid Series

3β-Acetoxy-B-nor-5β-cholestan-6-one (Ia) afforded only one isolatable oxime (IIa), while oximation of 3β, 17β-diacetoxy-B-nor-5β-androstan-6-one (Ib) yielded two isomeric oximes (IIb and IIIb). 7-Aza-5β-cholestan-3β-ol (VIa), 7-aza-5β-androstane-3β, 17β-diol (VIc), and 6-aza-5β-androstane-3β, 17β-diol (VIIc) were synthesized by Beckmann rearrangement of these oximes, followed by reduction with lithium aluminium hydride. The structure of the aza-steroids were established by conversion of the intermediate lactams (IVa, b) into the lactones (IXa, b), prepared from the 3β-acetoxy-B-nor-6-oxo-5β-steroids (Ia, b) by Baeyer Villiger reaction.     

Is L-gulonolactone-oxidase the only enzyme missing in animals subject to scurvy?

Evidence has recently appeared implicating an unusual microsomal D-glucuronolactone reductase, which requires carbonyl reagents for activity, in the biosynthesis of ascorbic acid. It was also shown that this microsomal enzyme activity was missing in guinea pigs and primates suggesting that L-gulonolactone oxidase deficiency was not the only defect in animals subject to scurvy. However, we have shown that highly purified L-glulonolactone oxidase catalyzes the conversion of the oxime and semicarbazone of D-glucuronolactone to the corresponding ascorbic acid derivative. There is, therefore, no need to propose a second pathway to ascorbic acid, nor is there evidence for more than the one enzyme defect in scurvy-prone animals.     

Continuing efforts on the improvement of Beckmann rearrangement of indanone oxime

Continuing search for beneficial additive toward the Beckmann rearrangement (BR) of indanone oxime has revealed that common Lewis acid catalyst in methanesulfonyl chloride (MsCl) showed increasing efficiency in this ionic rearrangement. The new protocol with MsCl is superior to the classical phosphorus-based methods such as PPA and Eaton reagent, especially in the reaction of indanone oximes.     

The biosynthesis of cyanogenic glucosides in higher plants. Identification of three hydroxylation steps in the biosynthesis of dhurrin in Sorghum bicolor (L.) Moench and the involvement of 1-ACI-nitro-2-(p-hydroxyphenyl)ethane as an intermediate

N-Hydroxytyrosine, (E)- and (Z)-p-hydroxyphenyl-acetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile are established intermediates in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin (Halkier, B. A., Olsen, C. E., and M?ller, B. L. (1989) J. Biol. Chem. 264, 19487-19494. Simultaneous measurements of oxygen consumption and biosynthetic activity using a microsomal enzyme system isolated from etiolated sorghum seedlings demonstrate a requirement for three oxygen molecules in the conversion of tyrosine to p-hydroxymandelonitrile. Two oxygen molecules are consumed in the conversion of tyrosine to (E)-p-hydroxyphenylacetaldehyde oxime, indicating the existence of a previously undetected hydroxylation step in addition to that resulting in the formation of N-hydroxytyrosine. Radioactively labeled 1-nitro-2-(p-hydroxyphenyl)ethane was chemically synthesized and tested as a possible intermediate. Biosynthetic experiments demonstrate that the microsomal enzyme system metabolizes the nitro compound to the subsequent intermediates in dhurrin synthesis (Km = 0.05 mM; Vmax = 14 nmol/mg of protein/h). Low amounts of 1-nitro-2-(p-hydroxyphenyl)ethane are produced in the microsomal reaction mixtures when tyrosine is used as substrate. These data support the involvement of 1-nitro-2-(p-hydroxyphenyl)ethane or more likely its aci-nitro tautomer as an intermediate between N-hydroxytyrosine and p-hydroxyphenylacetaldehyde oxime. The conversion of (E)-p-hydroxyphenylacetaldehydeoxime to p-hydroxymandelonitrile requires a single oxygen molecule. The oxygen molecule is utilized for hydroxylation of p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. This indicates that the conversion of p-hydroxyphenylacetaldehyde oxime into p-hydroxyphenylacetonitrile proceeds by a simple dehydration reaction.     

Conversion of indanone oximes into isocarbostyrils

New and effective method for the Beckmann rearrangement of indanone oxime mesylate is described, in which a selective and controlled production of the isomeric isocarbostyrils is achieved.     

Synthesis and anti-inflammatory activity of N-phthalimidomethyl 2,3-dideoxy- and 2,3-unsaturated glycosides

3,4,6-Tri-O-acetyl-D-galactal, 3,4,6-tri-O-acetyl-D-glucal and 3,6,2',3',4'6'-hexa-O-acetyl-D-lactal were reacted with N-hydroxymethylphthalimide and boron trifluoride etherate to produce the corresponding phthalimidomethyl unsaturated glycosides via Ferrier rearrangement. When the galactal derivative was used, a non-Ferrier rearrangement product was also isolated as a minor product under classical Ferrier conditions. Phthalimidomethyl deoxy glycosides were readily prepared by hydrogenation of the unsaturated glycosides. Following deacetylation, the anti-inflammatory activities of these compounds were tested on mice and three were found to possess potent activity compared to hydrocortisone sodium succinate (HSS).     

The biosynthesis of cyanogenic glucosides in higher plants. The (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime as intermediates in the biosynthesis of dhurrin in Sorghum bicolor (L.) Moench

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been studied with a microsomal preparation obtained from etiolated seedlings of sorghum. The biosynthetic pathway involves tyrosine, N-hydroxytyrosine, and p-hydroxyphenylacetaldehyde oxime as early intermediates (M?ller, B. L. and Conn, E. E. (1980) J. Biol. Chem. 254, 8575-8583). The use of deuterium-labeled tyrosine and mass spectrometric analyses demonstrate that the alpha-hydrogen atom of tyrosine is retained in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime. This excludes p-hydroxyphenylpyruvic acid oxime as intermediate in the pathway. A high pressure liquid chromatography method was developed to separate the (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime. The microsomal enzyme system was found to produce initially the (E)-isomer of p-hydroxyphenylacetaldehyde oxime. An isomerase then converts the (E)-isomer to the (Z)-isomer, which is the isomer preferentially utilized by the microsomal enzyme system in the subsequent biosynthetic reactions. The (E)-isomer produced in situ is more efficiently converted to the (Z)-isomer than exogenously added (E)-isomer and may thus be metabolically channeled.     

Prion phenomenon in medicine and biology

The data obtained suggest that the fatal changes in brain tissue associated with the prion diseases, are initiated by a conformational rearrangement of constitutively expressed cellular protein PrP. Possible mechanisms of such a conversion of this protein are discussed. Existence of the proteins with the prion properties in low eukaryotes may determine the unusual mechanisms of the "protein" inheritance. A new experimental model for studying the proteins with the prion properties in the yeast Saccharomyces cerevisiae, is described.     

Strategies for the analysis of chlorinated lipids in biological systems

Myeloperoxidase-derived HOCl reacts with the vinyl ether bond of plasmalogens yielding α-chlorofatty aldehydes. These chlorinated aldehydes can be purified using thin-layer chromatography, which is essential for subsequent analysis of extracts from some tissues such as myocardium. The α-chlorofatty aldehyde 2-chlorohexadecanal (2-ClHDA) is quantified after conversion to its pentafluorobenzyl oxime derivative using gas chromatography–mass spectrometry and negative-ion chemical ionization detection. 2-ClHDA accumulates in activated human neutrophils and monocytes, as well as in atherosclerotic lesions and infarcted myocardium. Metabolites of 2-ClHDA have also been identified, including the oxidation product, 2-chlorohexadecanoic acid (2-ClHA), and the reduction product, 2-chlorohexadecanol. 2-ClHA can be quantified using LC–MS with selected reaction monitoring (SRM) detection. 2-ClHA can be ω-oxidized by hepatocytes and subsequently β-oxidized from the ω-end, leading to the production of the dicarboxylic acid, 2-chloroadipic acid. This dicarboxylic acid is excreted in the urine and can also be quantified using LC–MS methods with SRM detection. Quantitative analyses of these novel chlorinated lipids are essential to identify the role of these lipids in leukocyte-mediated injury and disease.     

Oxime-metabolizing activity of liver aldehyde oxidase

Liver aldehyde oxidase in the presence of its electron donor exhibited a significant oxime-metabolizing activity toward some different types of oximes under anaerobic conditions. Acetophenone oxime and salicylaldoxime were exclusively converted to the corresponding oxo compounds, whereas benzamidoxime was converted to the corresponding ketimine. With d-camphor oxime, the formation of both the corresponding oxo compound and ketimine was observed. Stoichiometric studies showed that the formation of oxo compounds is accompanied by nearly equimolar ammonia. We propose a mechanism of oxime biotransformation that liver aldehyde oxidase catalyzes the reduction of oximes to the corresponding ketimines which in turn undergo, depending on their chemical stability, nonenzymatic hydrolysis to the corresponding oxo compounds and ammonia.     

Molecular Rearrangement of Longifolene by Arthrobacter ilicis T(2)

Arthrobacter ilicis T(2) brings about a unique type of cometabolic structural rearrangement of longifolene, a sesquiterpene, resulting in the formation of an acid. Infrared, nuclear magnetic resonance, mass spectrometry, and decoupling studies indicate that the acid product has a sativenelike structure, which is confirmed by conversion of the acid to its methyl ester and hydrocarbon.     

Catabolism of 3-Nitrophenol by Ralstonia eutropha JMP 134

Ralstonia eutropha JMP 134 utilizes 3-nitrophenol as the sole source of nitrogen, carbon, and energy. The entire catabolic pathway of 3-nitrophenol is chromosomally encoded. An initial NADPH-dependent reduction of 3-nitrophenol was found in cell extracts of strain JMP 134. By use of a partially purified 3-nitrophenol nitroreductase from 3-nitrophenol-grown cells, 3-hydroxylaminophenol was identified as the initial reduction product. Resting cells of R. eutropha JMP 134 metabolized 3-nitrophenol to N-acetylaminohydroquinone under anaerobic conditions. With cell extracts, 3-hydroxylaminophenol was converted into aminohydroquinone. This enzyme-mediated transformation corresponds to the acid-catalyzed Bamberger rearrangement. Enzymatic conversion of the analogous hydroxylaminobenzene yields a mixture of 2- and 4-aminophenol.     

Interesting reaction of the indanone oximes under Beckmann rearrangement conditions

Attempted Beckmann rearrangement of the 6-methoxyindanone oximes in conventional conditions resulted in the formation of the two kinds of unexpected products: 2-sulfonyloxyindanone and the dimeric product. Related rearrangement was also observed in the reaction with RhCl-trifluoromethansulfonic acid system.     

The action of alpha-ketoglutarate dehydrogenase on 4-chloronitrosobenzene: evidence for species-dependent differences in active site properties

The reaction of bovine (Bos taurus) and porcine (Sus scrufa) cardiac alpha-ketoglutarate dehydrogenase complex (alpha-KGD) with 4-chloronitrosobenzene (I) was shown to produce a hydroxamic acid (IV) and a product due to a Bamberger rearrangement as previously shown for Escherichia coli alpha-KGD. The conversion of I into an active site-bound electrophile was general among the three alpha-KGD enzymes tested, but quantitative differences in products and kinetics were shown. The reaction of I was specific for the resolved alpha-ketoglutarate decarboxylase subunit.