Synthetic Studies on Colchicine-related Tropolones

The condensation of 4-formyltropolone (II) and 3,4,5-trimethoxybenzoyl-acetate (III) afforded β-[1-hydroxy-3-oxo-3-(3,′4′5′-trimethoxyphenyl)-propy]-tropolone (IV) which was dehydrated to β-[3-oxo-3-(3′,4′,5′-trimethoxyphenyl)-1-propenyl]-tropolone (V). Catalytic hydrogenation of V gave β-[3-oxo-3(3′,4′,5′-trimethoxyphenyl)-propyl]-tropolone (VI), which was further reduced to β~[3-hydroxy-3-(3′,4′,5′-trimethoxyphenyl)-propyl]-tropolone (VII). The distillation of VII afforded finally β-3-(3′,4′,5′-trimethoxyphenyl)-2-propenyl]-tropolone (VIIIa). As the route to colchicine (I) from the tropolone (VIIIa) has already been exploited,¹⁾ this shows a total synthesis of colchicine from 4-formyltropolone.     

Syntheses and Biological Activities of Analogs of Abscisic Acid

Selenium dioxide oxidation of methyl α-ionylideneacetate (IIb) in ethanol afforded methyl 1′-and 4′-hydroxy-α-ionylideneacetate (IIIb and IV), methyl 3′-hydroxy-β-ionylideneacetate (V) and crude dihydroxy-ionylideneacetate (VI). The latter was oxidized with active manganese dioxide to give methyl abscisate (Ib). The growth and germination-inhibitory activity of compounds related to abscisic acid on Azuki bean seedlings and some species of seeds were examined.     

Chemical Studies on “Clover Sickness”

Two new isoflavonoids were isolated from red clover as germination inhibitors for the same plant and their structures were determined as a glucoside of biochanin A (7-d-β-glucosyl-5,7-dihydroxy-4′-methoxyisoflavone) (II) and its 5-malonate (I), respectively. Besides these compounds the following substances were also isolated as inhibitors: trifolirhizin (III), ononin (IV), daidzein (V) and its 7-glucoside (VI), formononetin (VII), genistein (VIII) and biochanin A (IX).     

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The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3′,5′-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP–cyclic adenosine 3′,5′-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP–CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.     

Synthesis of β-Tubanol Methylether

β-Tubanol methylether (IIb) was obtained by the Grignard reaction from methyl magnesium iodide and 5-methoxy-coumarin in boiling benzene. The preparation of 2-methyl-4-(2′-hydroxy-6′-methoxyphenyl) -buten-3-ol-2 (VI) and 2,2-dimethyl-7-methoxy-chromene-3 (IId) were also described.     

An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium

An H₂O₂-requiring enzyme system was found in the extracellular medium of ligninolytic cultures of Phanerochaete chrysosporium. The enzyme system generated ethylene from 2-keto-4-thiomethyl butyric acid (KTBA), and oxidized a variety of lignin model compounds including the diarylpropane 1-(4′-ethoxy-3′-methoxyphenyl) 1,3-dihydroxy-2-(4″-methoxyphenyl)propane (I), a β-ether dimer 1-(4′-ethoxy-3′-methoxyphenyl)glycerol-β-guaiacyl ether (IV) and an olefin 1-(4′-ethoxy-3′-methoxy-phenyl)1,2-propene (VI). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures. In addition, the enzyme system partially degraded ¹⁴C-ring labeled lignin. The enzyme was not found in high nitrogen (N) cultures, nor in cultures of a ligninolytic mutant strain which is incapable of metabolizing lignin.     

Studies on Abscisic Acid

Oxidation of 2-cis-α-ionylidene-ethanol (II) with active MnO₂ afforded a mixture of 2-cis and 2-trans-α-ionylideneacetaldehydes (III and IV). Reduction of methyl epoxy-α- and -β-ionylideneacetates (Vb, Xb XXIb and XXIIb) with LiAlH₄ gave the diols (VI, XI, XXIII and XXIV). The Wittig reaction of the hydroxyketones (XIII and XVIII) with carbethoxymethylenetriphenylphosphorane, followed by alkaline hydrolysis, yielded 5-(1′-and 2′-hydroxy-2′,6′,6′-trimethyl-1′-cyclohexyl)-3-methylpentadienoic acids (XIVa, XVa, XIXa and XXa). The reaction of α-cyclocitrylideneacetaldehyde (XXVII) and dihydro-α-ionone (XXXIII) with carbethoxymethylenetriphenylphosphorane afforded ethyl 3-demethyl-α-ionyli-deneacetate (XXVIIIb) and ethyl dihydro-α-ionylideneacetates (XXXIVb and XXXVb). Physiological activities of the above synthesized compounds on rice seedlings were examined.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

A New Synthesis of 2-Methyl-6-methyleneocta-2,7-dien-4-ol,A Component of the Pheromane of California Five-spined Ips

A key intermediate, 2-isocyano-3-hydroxybutyrate (III) was isolated from a reaction of isocyanoacetate (I) with acetaldehyde (II) in the presence of Et₃N. It was found that III was readily converted into 2-isocyanocrotonate (V) and 2-isocyano-2-(1′-hydroxyethyl)-3-hydroxybutyrate (VI) which are undesirable compounds for the synthesis of threonine. However, by use of a metal catalyst (e.g. NiCl₂ or PdCl₂), the isocyano-hydroxy compound (III) was selectively converted into 5-methyl-4-alkoxycarbonyl-2-oxazoline (IV) which is an important precursor of threonine. Furthermore, chemical properties of IV were examined; the results suggested that cis-oxazoline was relatively sensitive to acid, base and heat.

On the basis of these results, the reaction of I with II was carried out using Et₃N-PdCl₂ as a catalyst to obtain threo-threonine (85% purity) in a good yield (85%).     

Utilization of Bacterial Xanthine Oxidase for Inorganic Phosphorus Determination

Two host-specific phytotoxic metabolites, AK-toxin I and II, were isolated from a culture broth of Alternaria alternata Japanese pear pathotype, the fungus causing black spot disease of susceptible Japanese pear cultivars. From chemical, spectral and X-ray crystallographic data, AK-toxin I was characterized as 8-(2′S, 3′S)-2′-acetylamino-3′-methyl-3′-phenyl-propionyloxy]-(8R,9S)-9,10-epoxy-9-methyl-deca-(2E,4Z,6E)-trienoic acid. The structure of AK-toxin II was also assigned to be 3′-demethyl derivative of AK-toxin I by comparing the spectral data with those of AK-toxin I.     

Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.     

Immunohistochemical demonstration of the carbonic anhydrase isoenzymes I and II in pancreatic tumours

Summary The location of carbonic anhydrase (CA) isoenzymes I, II and VI in normal and neoplastic pancreatic tissue was studied using polyclonal antisera and the immunoperoxidase technique. Samples were obtained from patients with well-differentiated (n = 4), moderately differentiated (n = 1) and poorly differentiated (n = 4) ductal adenocarcinomas, cystadenocarcinoma (n = 2), adenosquamous carcinoma (n = 1), acinar adenocarcinoma (n = 1), gastrinoma (n = 3), insulinoma (n = 3) and glucagonoma (n = 1). The control specimens were from a patient with traumatic laceration of the pancreas. The normal and malignant endocrine tissue showed intense positive staining for CA I localized in the cells expressing glucagon. In the exocrine pancreatic tissue, CA II was detected in the normal and neoplastic ductal epithelium. No specific staining was detected with anti-CA VI serum in either normal or malignant tissue.     

Metabolism of a Non-phenolic β-O-4 Lignin Substructure Model Compound by Coriolus versicolor

A non-phenolic β-O-4 lignin substructure model, 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I), was metabolized by a ligninolytic culture of Coriolus versicolor. Based on the identification of the metabolic products (II~XI), the following reactions were found to occur in the culture; a) oxidation (III) and reduction (II) at the benzyl (Cα′) position of the substrate (I), b) β-ether cleavage to give arylglycerols (IV, V), and c) Cα-Cβ cleavage of the arylglycerols and/or arylglycerol moiety of the substrate (I). In addition, β-deoxy diol (VI) and γ-formylglycerol (VII) were obtained as degradation products from substrate (I).     

Syntheses and Configurational Analysis of Rotenoids,XIX The Total Synthesis of Natural Rotenone

d,l-Derrisic acid (IIa, R′: H) was synthesized from d,l-hydroxy dihydrotubanol (Ve) by Hoesch condensation with a nitrile (VI). The possible optical resolution of d,l-IIa was demonstrated by a conventional “reversed resolution” method.

This communication and previous works constitute the first total synthesis of natural rotenone (Ia).     

The intracellular blue pigment of Pseudomonas lemonnieri

Summary The large-scale production, isolation, and purification are described of the blue insoluble intracellular pigment of the bacterium Pseudomonas lemonieri. The pigment, C₂₆H₃₇N₅O₆, occurs in the cells as a salt (cation unknown) of 6-octanoylamino-3-hydroxy-2-aza-benzoquinone-(1,4)-4-[5-octanoylamino-2,6-dihydroxy-pyridyl-(3)-imide] (I). Nitric acid oxidation of pigment I yields IV, 6-octanoylamino-3-hydroxy-2-aza-benzoquinone-(1,4). Further hydrolysis of IV splits off n-octanoic acid, which is free of homologues. The structures given for the pigment and its degradation products have been proven by identification with authentic preparations.Although they have different chromophores, the pigment (I) of Pseudomonas lemonnieri and N,N-dioctanoyl-indigoidine (VI) nevertheless resemble one another in IR-absorbances, NMR-spectra, and chromatographic behavior, because of homogeneous functional groups and ring structures. I and VI are indeed chemically related, as can be seen from the facts that aminocitrazinic acid is a common starting material for the in vitro syntheses of both compounds, and that the diazaindophenol (I) can be converted to the diaza-diphenoquinone (VI) by hydrogenation and subsequent autoxidation.Dedicated with devotion and admiration to Professor C. B. Van Niel on his seventieth birthday.     

Formation of Glucose Isomerase by Streptomyces sp.

Sophoradin (I) [2′,4,4′-trihydroxy-3,3′,5-tris(3-methyl-2-butenyl)chalcone] which had been isolated from “Guang-Dou-Gen” (the root of Sophora subprostrata Chun et T. Chen) was synthesized through Claisen rearrangement. The reaction of p-hydroxybenzaldehyde and 3-chloro-3-methyl-1-butyne (III) gave 4-(1,1-dimethylpropargyloxy)benzaldehyde (VIII), which was catalytically hydrogenated over Lindlar catalyst to afford 4-(1,1-dimethylallyloxy)benzaldehyde (IX). IX was converted to 4-hydroxy-3-(3-methyl-2-butenyl)benzaldehyde (X) by Claisen rearrangement. The reaction of X and III gave 3-(3-methyl-2-butenyl)-4-(1,1-dimethylpropargyloxy)benzaldehyde (XI). Condensation of 2-hydroxy-4-(1,1-dimethylpropargyloxy)acetophenone (IV) and XI in alkaline solution gave a chalcone (XIII), which was catalytically hydrogenated over Lindlar catalyst to give 2′-hydroxy-4,4′-bis(1,-dimethylallyloxy)-3-(3-methyl-2-butenyl)chalcone (XIV). XIV was converted to I by Claisen rearrangement.     

Leaf Alcohol

The diethylamine-catalyzed aldol condensation of E-2-hexenal yielded a mixture of 2-E,4-E,6-E- (IV-a) and 2-E,4-Z,6-E-4-ethyldeca-2,4,6-triene-1-al (IV-b). Structual and geometrical elucidation of both alcohols were made by means of spectral evidence as well as by the catalytic hydrogenation leading to the same 4-ethyldecanol (VI). The “b-peak substance” detected in the leaf alcohol reaction products was proved to be identical with 4-ethyldecanol (VI). The treatment of the trienal containing the central Z-double bond with sodium under the leaf alcohol reaction condition failed to afford ethyl-propyl-benzyl alcohol, but gave 4-ethyldecanol (VI). This result safely excludes the operation of the previously suspected valence tautomerism (Cope rearrangement) in the leaf alcohol reaction, and accounts for the pathway of the formation of (VI).     

Chemical Constituents of a Heat-dried Chinese Mushroom,Hohenbuehelia serotina

Several compounds were isolated from a Chinese mushroom, Huangmo, the heat-dried fruiting body of Hohenbuehelia serotina. They were identified as linoleic acid (I), hexadecanoic acid (II), β-sitosterol (IV), benzoic acid (V), D-mannitol (VI), sucrose (VII), and L-rhamnose (VIII). In addition, six acidic substances were identified. (Table I). Also, ethyl linoleate, hexadecanoic acid, and 9,12-octadecadienoic acid (Z-Z) ethyl esters, IV, V, and VI were identified for the first time from this mushroom.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

Rational Approaches to the Design of Mechanism-Based Inhibitors of S-Adenosylhomocysteine Hydrolase

Abstract

Crucial to the rational design of inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase was the elucidation of its mechanism of catalysis by Palmer and Abeles (J. Biol. Chem. 254, 1217–1226, 1979). This mechanism involves an NAD⁺-dependent oxidation (oxidative activity) of the 3′-hydroxyl group of AdoHcy followed by elimination of homocysteine (Hcy) to form 4′,5′-didehydro-3′-keto-Ado. Addition of water at the 5′-position (hydrolytic activity) of this tightly bound intermediate followed by an NADH-dependent reduction results in the formation of adenosine (Ado). Many inhibitors of this enzyme have been shown to serve as substrates [e.g., 9-(trans-2-trans-3-dihydroxycyclopent-4-en-1-yl)adenine, DHCeA)] for the oxidative activity of AdoHcy hydrolase, affording the 3′-keto-derivative (e.g., 3′-keto-DHCeA), which is tightly bound to the enzyme, and converting the enzyme from its active form (NAD⁺) to its inactive form (NADH) (Type I mechanism-based inhibitors; Wolfe and Borchardt, J. Med. Chem. 34, 1521–1530, 1991). More recently, substrates [e.g., (E)-5.,6′-didehydro-6′-deoxy-6′-fluorohomoadenosine, EDDFHA] for the hydrolytic activity of AdoHcy hydrolase have been identified by our laboratories. Identification of hydrolytic substrates affords a new strategy for the design of more potent and more specific inhibitors of AdoHcy hydrolase.