Stopped-flow Measurement of Reaction Rate of Triose Reductone with 2,6-Dichlorophenolindophenol and Its Application to the Determination of Triose Reductone

The reduction of 2, 6-dichlorophenolindophenol by triose reductone was studied in a wide pH range (pH 2~11), using a stopped-flow apparatus. The apparent first-order rate constant, k_app, and the second-order rate constant, k, were obtained. The k-pH profile resembles that of l-ascorbic acid, though the rate of l-ascorbic acid is considerably larger than that of triose reductone. Determination of triose reductone by stopped-flow method is possible, even in the presence of various organic substances.     

Synthetic and Antiviral Studies of Carboacyclic 6- and 2,6-Substituted Purine Nucleosides

Abstract

Chiral purine derivatives containing a carboacyclic chain mimicking the ribose ring, but lacking the C(3′)-C(4′) bond have been prepared from (2S, 3R)-3-amino-1,2,6-tribenzyloxyhexane 15. The synthesis of this amine via an hex-2-enopyranoside utilizes the absolute configuration defined by carbons 4 and 5 of D-glucose. None of these compounds exhibited any antiviral activity against HIV.     

Norepinephrine Metabolism in Man Using Deuterium Labelling: The Conversion of 4-Hydroxy-3-Methoxyphenylglycol to 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study the disposition of peripherally administered HMPG. Five healthy men were given an intravenous pulse dose of 4.3 μmol of labelled HMPG and subsequent plasma and urine levels of endogenous and labelled HMPG as well as those of 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) were determined by gas chromatography-mass spectrometry, using selected ion detection. Approximately 40% of the injected amount of deuterium-labelled HMPG was recovered in the urine as HMMA and another 40% was eliminated as HMPG conjugates. Thus, the HMPG formed from norepinephrine either in the central or peripheral nervous system undergoes both conjugation and extensive oxidation.     

Syntheses of 2-Substituted 6/7-Methoxy-l,4-benzodioxan-7/6-carbaldehydes

Five 2-substituted 6/7-methoxy-l,4-benzodioxan-7/6-carbaldehydes and 6-methoxy-l,4-benzodioxan-7-carbaldehyde available for the syntheses of insecticidal neolignan analogs were prepared from 4/3-benzyloxy-3/4-hydroxybenzaldehydes and l,4-benzodioxan-6-carbaldehyde, respectively.     

Production of Ribotides of 4-Hydroxy- and 4-Aminopyrazolo[3, 4-d]pyrimidine by Brevibacterium ammoniagenes

Brevibacterium ammoniagenes ATCC 6872 was previously reported to accumulate large amounts of IMP, AMP, ADP, ATP, GMP, GDP and GTP from the corresponding purine bases. The organism was also reported to convert various derivatives of purine and 8-azapurine to the corresponding ribotides.

Using the similar process, ribotidation of pyrazolo[3, 4-d]pyrimidines was attempted, and it was found that the same organism was able to produce remarkable amounts of 4-hydroxy-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate (HPP-RP) from 4-hydroxypyrazolo[3, 4-d]pyrimidine (HPP, allopurinol) and 4-amino-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate and 5′-diphosphate from 4-amino-pyrazolo[3, 4-d]pyrimidine.

The crystals of HPP-RP (Na-salt) were isolated from the cultured broth of Br. ammoniagenes incubated with HPP, and characterized based on UV-spectra, IR-spectrum, NMR and others.

It was also found that HPP-RP was converted to the corresponding riboside by hydrolysis in aqueous solution (pH 4.0 ~ 9.0) for 6 hr at 140°C. The hydrolysis of HPP-RP was also accomplished with various organisms.     

Further Studies on 4-Hydroxy-3-Methoxyphenylglycol Oxidation in Humans: Effect of Pool Expansion and Stereochemistry

The in vivo oxidation of the norepinephrine metabolite 4-hydroxy-3-methoxyphenylglycol (HMPG) to 4-hydroxy-3-methoxymandelic acid was studied in man with two different doses of deuterium-labeled HMPG and a tracer dose of [14C]HMPG. HMPG oxidation appeared to be dose-dependent with an oxidation of 62-70% for doses below or equal to 2.2 mumol. With the use of a capillary column coated with an optically active phase (Chirasil-Val) and gas chromatography mass-spectrometry the human urinary excretions of the two stereoisomers of deuterium-labelled HMPG (free + conjugates) were found to be equal.     

Norepinephrine Metabolism in Man Using Deuterium Labelling: Origin of 4-Hydroxy-3-Methoxymandelic Acid

A double isotope labelling technique was used to simultaneously determine the in vivo turnover rates of 4-hydroxy-3-methoxyphenylglycol (HMPG) and 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) and the rate of HMPG oxidation to HMMA. Six healthy men were given intravenous injections of [2H3]HMPG and [2H6]HMMA and their plasma and urine samples analysed by gas chromatography--mass spectrometry (GC/MS) for the protium and deuterium species. HMPG and HMMA production rates were calculated by isotope dilution. The rate of HMPG oxidation to HMMA was obtained from the fraction of [2H3]HMPG recovered as [2H3]HMMA. The results showed that the entire production of HMMA, 1.11 +/- 0.21 mumol/h (mean +/- SE), could be accounted for by oxidation of HMPG, 1.49 +/- 0.31 mumol/h. In another experiment designed to avoid expansion of the HMPG body pool, a tracer dose of [14C]HMPG was given to the same subjects. The levels of [14C]HMPG and [14C]HMMA were measured in urine after extraction and separation by thin layer chromatography. Urinary excretion of endogenous HMPG and HMMA was determined by GC/MS. The results showed that the endogenous HMMA fraction of the total HMPG and HMMA urinary excretion rate, 0.57 +/- 0.04, was the same as the fraction of [14C]HMPG oxidized to [14C]HMMA, 0.62 +/- 0.01. Thus, HMPG is the main intermediate in the metabolic conversion of norepinephrine and epinephrine to HMMA in man.     

Norepinephrine Metabolism in Man Using Deuterium Labeling: Turnover of 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 μmol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography-mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 × 10% (mean ± SD). No conversion of HMMA t o 4-hydroxy-3-methoxyphenyl glycol (HMPG) occurred. The biological half-life of HMMA was 0.54 ± 0.22 h. The apparent volume of distribution was 0.36 ± 0.11 L/kg. The production rate or body turnover was 1.27 ± 0.51 μmol HMM/h and urinary excretion rate was 0.82 ± 0.22 μmol/h. These results show that HMMA is turning over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.     

Production of 3-Methyl-6-Methoxy-8-Hydroxy-3,4-Dihydroisocoumarin by Aspergillus caespitosus

Aspergillus caepitosus Raper and Thom cultured on autoclaved cracked, yellow field corn produced 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin which was extracted and then purified by silica gel columns. The physical and spectral properties of the colorless crystals from methylene chloride:hexane were consistent with those published for 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin. Yield of high purity 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin was about 140 mg of corn per kg.     

Formation of 3-Keto-4-deoxypentosone and 3-Hydroxy-2-pyrone by the Degradation of Dehydro-l-ascorbic Acid

A crystalline phenylhydrazone was obtained when a heated solution of dehydro-l-ascorbic acid (DHA) was treated with phenylhydrazine-HCl. Its molecular formula was C₁₇H₁₈N₄O₂, and the structure was determined to be 1,2-bis(phenylhydrazone) of 3-keto-4-deoxypentosone, a new tricarbonyl compound which was considered to be one of the possible intermediates of the browning reaction of DHA. 3-Hydroxy-2-pyrone was also isolated from the ether extract of the heated DHA solution as a main aroma compound produced from DHA. Possible formation mechanisms of these compounds were discussed.     

Determination of Site-Specific Modifications of Glucose-6-Phosphate Dehydrogenase by 4-Hydroxy-2-Nonenal Using Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry

Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1, 4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1, 4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.     

Simulation Studies of Vestibular Macular Afferent-Discharge Patterns Using a New, Quasi-3-D Finite Volume Method

A quasi-three-dimensional finite-volume numerical simulator was developed to study passive voltage spread in vestibular macular afferents. The method, borrowed from computational fluid dynamics, discretizes events transpiring in small volumes over time. The afferent simulated had three calyces with processes. The number of processes and synapses, and direction and timing of synapse activation, were varied. Simultaneous synapse activation resulted in shortest latency, while directional activation (proximal to distal and distal to proximal) yielded most regular discharges. Color-coded visualizations showed that the simulator discretized events and demonstrated that discharge produced a distal spread of voltage from the spike initiator into the ending. The simulations indicate that directional input, morphology, and timing of synapse activation can affect discharge properties, as must also distal spread of voltage from the spike initiator. The finite volume method has generality and can be applied to more complex neurons to explore discrete synaptic effects in four dimensions.     

Induction of a Thionin, the Jasmonate-Induced 6 kDa Protein of Barley by 2,6-Dechloroisonicotinic acid

The systemic acquired resistance (SAR) was studied in barley to find out specific molecular markers for this type of resistance. Such markers may serve as diagnostic tools to indicate the defense-status of a plant and, additionally, may be used to identify new resistance-inducing compounds in broad screening experiments. Upon treatment of barley leaves with the resistance-inducing compound 2,6-dichloroisomicotinic acid (INA) we found, in addition to a yet unidentified basic protein of 45 kDa (designated BIR-1) induction of a specific 6kDa polypeptide. Using Northern-and Western blot analysis this small polypeptide was identified as JIP-6, a known member of the jasmonate-induced protein (JIP)-family of barley that was previously demonstrated to be a leaf thionin (ANDRESEN et al. 1992).     

Mechanism of the 6-Hydroxy-3-succinoyl-pyridine 3-Monooxygenase Flavoprotein from Pseudomonas putida S16

6-Hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB), a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, catalyzes pyridine-ring β-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine. Here, we generated His₆-tagged HspB in Escherichia coli, characterized the properties of the recombinant enzyme, and investigated its mechanism of catalysis. In contrast to conclusions reported previously, the second product of the HspB reaction was shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H₂O. Phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB. The reduction of HspB (with maxima at 375 and 460 nm, and a shoulder at 485 nm) by NADH was followed by stopped-flow spectroscopy, and the rate constant for reduction was shown to be stimulated by HSP. Reduced HspB reacts with oxygen to form a C_(4a)-(hydro)peroxyflavin intermediate with an absorbance maximum at ∼400 nm within the first few milliseconds before converting to the oxidized flavoenzyme species. The formed C_(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that hydrolyzes to the products 2,5-dihydroxypyridine and succinate. The investigation on the catalytic mechanism of a flavoprotein pyridine-ring β-position hydroxylase provides useful information for the biosynthesis of pyridine derivatives.     

Syntheses of 2-Isopropyl-4,5-dihydrothiazole and 6-Hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus

Syntheses of 2-isopropyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus, were achieved to provide sufficient amounts of samples for biological studies.     

Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate by Fungi

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).     

A Novel Gene, Encoding 6-Hydroxy-3-Succinoylpyridine Hydroxylase, Involved in Nicotine Degradation by Pseudomonas putida Strain S16

Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a way similar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine (HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are two important precursors for drug syntheses. However, there is little information on the molecular mechanism for nicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bp gene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine, HSP, and DHP were identified from resting cell reactions of the transformant containing the gene cluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonas putida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directly to DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa) is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acid shows no similarity with any protein of known function.