Proposed mechanism for the reaction catalyzed by a diterpene cyclase,aphidicolan-16beta-ol synthase: experimental results on biomimetic cyclization and examination of the cyclization pathway by ab initio calculations

To examine the mechanism of the cyclization reaction catalyzed by aphidicolan-16beta-ol synthase (ACS), which is a key enzyme in the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha, skeletal rearrangement of 2a and biomimetic cyclization of 4b were employed. The structures of the reaction products, which reflect penultimate cation intermediates, allowed us to propose a detailed reaction pathway for the Lewis acid-catalyzed cyclizations and rearrangements. Isolation of these products in an aphidicolin-producing fungus led us to speculate that the mechanism of the ACS-catalyzed cyclization reaction is the same as that of a nonenzymatic reaction. Ab initio calculations of the acid-catalyzed reaction intermediates and the transition states indicate that the overall reaction catalyzed by ACS is an exothermic process though the reaction proceeds via an energetically disfavored secondary cation-like transition state. In conjunction with the solvent effect in the acid-catalyzed reactions, this indicates that the actual role of ACS is to provide a template which enforces conformations of the intermediate cations leading to the productive cyclization although it has been believed that the cation-pi interaction between cation intermediates and aromatic amino acid residues in the active site is important for the enzymatic catalysis. This study provided important information on the role of various cationic species, especially secondary cation-like structures, in both nonenzymatic and enzymatic reactions.     

Application of europium(III) extraction with organophosphinic acid to liquid membrane transport

The extraction behavior of Eu(III) has been studied using di(2,4,4-trimethylpentyl)phosphinic acid (DTMPPA, HA) in kerosene. Europium was extracted as Eu(HA₂)₃ with the extraction constant of 2.0·10^–3. This extraction system was applied to the transport of Eu(III) across a DTMPPA liquid membrane supported on porous polytetrafluoroethylene. Europium was quantitatively moved through the liquid membrane containing 0.1M (HA)₂ as a mobile carrier from the feed solution of pH above 3 into the product solution of 0.1M HNO₃, yielding a concentration factor of ten. The transport rate increased with increasing pH and DTMPPA concentration.     

A temperature study on critical micellization concentration (CMC), solubility, and degree of counterion binding of α-sulfonatomyristic acid methyl ester in water by electroconductivity measurements

 For a sodium salt of α-sulfonatomyristic acid methyl ester (14SFNa), one of the α-SFMe series surfactants, critical micellization concentration (CMC), solubility and degree of counterion binding (β) were determined by means of electrocon-ductivity measurements at different temperatures (at every 5 °C) ranging from 15 to 50 °C. The phase diagram of 14SFNa in pure water was constructed from the CMC- and solubility-temperature data, in which the Krafft temperature (critical solution temperature) was found around 0 °C. The changes in the Gibbs energy, ΔG ⁰ _m, enthalpy, ΔH ⁰ _m, and entropy, ΔS ⁰ _m, upon micelle formation as a function of temperature were evaluated taking βvalues into calculation. Received: 28 August 1996 Accepted: 5 November 1996     

Dynamic covalent approach to [2]- and [3]roxtanes by utilizing a reversible thiol-disulfide interchange reaction

A dynamic covalent approach to disulfide-containing [2]- and [3]rotaxanes is described. Symmetrical dumbbell-shaped compounds with two secondary ammonium centers and a central located disulfide bond were synthesized as components of rotaxanes. The rotaxanes were synthesized from the dumbbell-shaped compounds and dibenzo-[24]crown-8 (DB24C8) with catalysis by benzenethiol. The yields of isolated rotaxanes reached about 90 % under optimized conditions. A kinetic study on the reaction forming [2]rotaxane 2 a and [3]rotaxane 3 a suggested a plausible reaction mechanism comprising several steps, including 1) initiation, 2) [2]rotaxane formation, and 3) [3]rotaxane formation. The whole reaction was found to be reversible in the presence of thiols, and thermodynamic control over product distribution was thus possible by varying the temperature, solvent, initial ratio of substrates, and concentration. The steric bulk of the end-capping groups had almost no influence on rotaxane yields, but the structure of the thiol was crucial for reaction rates. Amines and phosphines were also effective as catalysts. The structural characterization of the rotaxanes included an X-ray crystallographic study on [3]rotaxane 3 a.     

Multilayer vesicles and vesicle clusters formed by the fullerene-based surfactant C60(CH3)5K

The self-assembly behavior of a fullerene-based surfactant, C60(CH3)5K, in water was studied using a combination of static and dynamic light scattering, as well as transmission electron microscopy, and compared to that of the compound C60(C6H5)5K. Both fullerene surfactant systems spontaneously assemble into large vesicles consisting of closed spherical shells formed by bilayers, with critical aggregation concentrations (CAC) lower than 10(-6) g ml(-1). At low concentrations, the aggregate sizes of C60(CH3)5K (radius R approximately 26.8 nm) and C60(C6H5)5K (R approximately 17.0 nm) were found to be substantially different from each other, showing that the change of the substituents surrounding the polar cyclopentadienide head group makes it possible to control the size of the resulting aggregates. Furthermore, the C60(CH3)5K vesicles were found to exist in two qualitatively different types of aggregation with a critical reaggregation concentration (CRC) located at 3.30 x 10(-6) g ml(-1). Above the CRC, larger aggregates were observed (R approximately 37.6 nm), showing a more complex form of supramolecular aggregation, e.g., in terms of multi-bilayer vesicles and/or of clusters of bilayer vesicles.     

Analysis of Dns-amino acids by liquid chromatography. I. Selection of optimum mobile phase composition for separation of Dns-amino acids on polyvinyl acetate gel.

The separation of a mixture of ten Dns-amino acids (Gns-Gly, -Ala, -Val, -Leu, -Pro, -Hypro, -Met, -Ser, -Asn and -Gln) was carried out by liquid chromatography by using macroreticular polyvinyl acetate gel as a packing material. Different mobile phase systems were investigated, based mainly on mixtures of n-hexane with ethanol, methanol, chloroform, acetone, methyl ethyl ketone, ethyl acetate, dioxane and tetrahydrofuran. The solvent composition was fixed so as to elute all of the components of the sample mixture in a practical period of 2 h. Satisfactory separation of the ten components was obtained with the n-hexane-ethanol (90:10) system. The presence of methanol as a modifier in the n-hexane was effective in reducing the elution time, but the separation was not as satisfactory. Chloroform or dioxane was useful only for the separation of Ser, Asn and Gln. Acetone, methyl ethyl ketone, ethyl acetate and tetrahydrofuran were not suitable for practical separations of Dns-amino acids.     

Application and limitation of a mathematical distortion model of an absorbance signal in electrothermal atomic absorption spectrometry

A distortion model of instrumental function is described, based on the response signal. Its limitations are investigated by using quasi-signals generated by an oscillator and a moving carbon rod.     

Isomerization polymerization of β-propiolactones carrying side-chain ester groups

Based on our recent discovery of the isomerization polymerization of β-(2-acetoxyethyl)-β-propiolactone into poly-δ-ester,^1,2 we examined the generality of this phenomenon by using two related monomers. The catalysts were (EtAlO)_n and Et(ZnO)₂ZnEt. The side-chains in the monomers selected were the (CH₃)₂CHCOO? CH₂CH₂? (2) and (CH₃)CICHCOO? CH₂CH₂? (3) groups in which steric effects are almost identical but electronic effects are in opposition. The monomers yielded isomerized poly-δ-ester units, depending on the terminal substituent groups in the side-chain. These observations can be interpreted with the bicyclic intermediate proposed in the earlier work. Monomer (2) was reactive and produced a poly-δ-ester structure most readily, probably because of the higher electron density at the side-chain ester group which coordinated with the catalyst. In contrast, monomer (3) was less reactive, and the probability of isomerization was the lowest, i.e., the electron deficient side-chain ester group apparently interfered with the formation of the intermediate, especially in the Zn-catalysis. Equibinary random copolymers were prepared from (2) and (3) according to the catalyst and polymerization conditions chosen.     

Thermal decomposition of the solvent-extracted chloro complexes of vanadium(IV) with trioctylamine and trioctylmethylammonium chloride

The complexes (R₃NHVO(OH)Cl₂)₂ and (R₃RNVO(OH)Cl₂)₂ prepared by drying in vacuo the organic solutions from the extraction into benzene of aqueous vanadyl chloride solution with trioctylamine (TOA, R₃N) and trioctylmethylammonium chloride (TOMAC, R₃RNCl), were studied by thermogravimetry and differential thermal analysis under an atmosphere of nitrogen, and the products of their thermal decomposition, such as volatile matter and residues, by gas chromatography. X-ray diffraction and infrared spectroscopy. It was found that the complexes with TOA and TOMAC thermally decompose to V₂O₃ at 200–300° by cracking of the compounds R₃NHVO(OH)Cl₂ and R₃RNVO(OH)Cl₂ formed via dissociation of the complexes at about 150°.

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Zusammenfassung Die Komplexe (R₃NHVO(OH)Cl₂)₂ und (R₃RNVO(OH)Cl₂)₂ wurden hergestellt, indem benzolische und wässrige Vanadylchlorid-Lösungen mit Trioctylamin (TOA, R₃N) und Trioctylmethylammoniumchlorid (TOMAC, R₃RNCl) extrahiert und die erhaltenen Lösungen im Vakuum eingedampft wurden. Die Komplexen wurden in Stickstoffatmosphäre mittels TG und DTA untersucht und die thermischen Zersetzungsprodukte, und zwar sowohl die flüchtigen Verbindungen als auch die Rückstände, gaschromatographisch bzw. röntgendiffraktometrisch und IR-spektroskopisch bestimmt. Es wurde festgestellt, daß die Komplexe mit TOA und TOMAC bei 150° in die Verbindungen R₃NHVO(OH)Cl₂ und R₃RNVO(OH)Cl₂ dissoziieren, die bei 200–300° thermisch zu V₂O₃ zersetzt werden.

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(R₃NHVO(OH)Cl₂)₂ (R₃RNVO(OH)Cl₂)₂ (R₃,) ( , R₃RNCl). , , . , 200–300° V₂₃ R₃NV()l₂ R₃RNVO(OH)Cl₂, 150°.

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We wish to thank the Koei Chemical Co. Ltd. for samples of TOA and TOMAC.