Purification of enzymes by high-speed gel filtration on TSK-GEL SW columns

The purification of enzymes was investigated by high-speed gel filtration on TSK-GEL G3000SWG columns packed with porous silica gel deactivated by chemically bonded hydrophilic compounds. Crude β-galactosidase from bacterial cells and commercial urease were purified ca. 15-fold in a single gel filtration. These enzymes were eluted within an hour from the column and the recoveries of enzymatic activity were almost 100% although the operation was carried out at room temperature (22°). Samples up to 100 mg could be applied to the column without loss of separation efficiency.     

High modulus polyimide/polyimide molecular composite films that utilize acetylene unit as a crosslink site

Polyimide/polyimide molecular composite (MC) films comprised of a rigid polyimide derived from biphenyltetracarboxylic dianhydride (BPDA) and p-phenylenediamine (PDA) and a flexible polyimide derived from BPDA and bis (3,3'-diaminodiphenyl) acetylene (intA) and/or oxydianiline (ODA) were prepared by blending the polyamic acid solutions in 7 : 3 weight ratio, and then imidizing the blend films. Acetylene content in the flexible polyimide backbone was controlled by the ratio of intA and ODA. Cold-drawing of the blend polyamic acid films, followed by imidization, gives high modulus polyimide/polyimide MC films. The modulus of the MC films increased almost linearly with the draw ratio, reaching 25.5 GPa for the 40% drawn film. Acetylene groups in the flexible polyimide can be thermally cured to crosslink. The onset of exotherm appeared at 340°C on DSC, reaching maximum at 398°C. After the thermal crosslinking, the MC films maintained the high modulus, though elongation became small. Taking advantage of the crosslinkable acetylene units, two MC films were laminated and processed at 400°C for 20 min under 100 kg/cm² to give a good-quality laminate film. The interface of the two films was strongly bonded through the crosslinking of acetylene groups. Laminate films maintained the high modulus afforded by the cold-drawing. © 1994 John Wiley & Sons, Inc.     

Highly regio- and stereoselective hydrometallation reactions of fluorine-containing internal alkynes: Novel approaches to fluoroalkylated alkenes

Hydroalumination, hydrocupration, and hydroboration reactions of various fluorine-containing alkynes were investigated. The alkyne reacted smoothly with 2.0 equiv. of Red-Al at −78 °C to give the hydroaluminated adduct in a highly regio- and stereoselective manner, which was treated with iodine, the corresponding vinyliodide being produced in moderate yield. Hydrocupration of the alkynes also took place, but the resulting vinylmetal reacted with various electrophiles sluggishly. In sharp contrast, the reaction with dicyclohexylborane proceeded smoothly to afford the cis-addition products preferentially, which were subjected to Suzuki-Miyaura cross-coupling reaction, leading to trisubstituted alkenes in high yields.     

Highly Selective Separation of Rhodopsin from Bovine Rod Outer Segment Membranes Using Combination of Divalent Cation and Alkyl(thio)glucoside

The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophilic–lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent-to-membrane ratio. This allows remarkable purification of rhodopsin by a single-step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low-speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A₂₈₀/A₅₀₀ value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB-series cations (Zn²⁺ and Cd²⁺) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.     

Chemistry of the neopolyoxins,pyrimidine and imidazoline nucleoside peptide antibiotics

New chitin synthetase inhibitors, neopolyoxins A, B, and C were isolated from the culture filtrate of Streptomyces cacaoi subsp. asoensis. Their absolute structures have been established on the basis of chemical and spectroscopic evidence. They are structural analogs of the polyoxins. As a nucleobase, neopolyoxin C possesses uracil, while neopolyoxins A and B contain the imidazoline moiety. A ring contraction reaction of pyrimidine nucleoside into imidazoline nucleoside played a key role in the structure determination of the nucleoside moiety. A similar transformation was suggested for the biosynthesis of neopolyoxins A and B.     

Coating and water permeation properties of SiO2 thin films prepared by the sol-gel method on nylon-6 substrates

Nylon-6 substrates were coated with SiO₂ thin films by the sol-gel method and their water permeability coefficient was evaluated. Methyltriethoxysilane (MTES) and tetraethoxysilane (TEOS) were used as starting materials. The addition of MTES to TEOS has enabled the formation of crack-free thin films on the substrates. The thin films strongly adhered to the substrates. The water permeability coefficients of nylon-6 substrates coated with these thin films decreased with the increase in the ratio of TEOS to the total alkoxides. The pretreatment of the nylon-6 substrates with -aminopropyltriethoxysilane was found to be effective to suppress the water permeability. The water permeability was suppressed by about 40% under the optimal condition.     

Nitrosation of Phenolic Substrates under Mildly Basic Conditions: Selective Preparation of p-Quinone Monooximes and Their Antiviral Activities

Nitrosation of 3-methoxyphenol and 1-naphthol were examined under both acidic (NaNO(2)-EtCO(2)H-H(2)O) and basic (i-AmNO(2)-K(2)CO(3)-DMF) conditions. Acidic nitrosations afforded ortho-directed products, whereas para-directed nitrosations were observed under basic conditions to yield p-quinone monooximes. The basic para-directed nitrosation was further examined using 15 phenols, two naphthols, and four phenolic heterocyclics. A one-pot operation of the basic nitrosation followed by methylation with dimethyl sulfate gave the corresponding methyl ethers in high yield. Two p-quinone monooximes derived from 3-methoxyphenol and 8-hydroxyquinoline showed a moderate activity against HSV-1, and the latter oxime was also effective against HSV-2. On the other hand, p-quinone monooximes derived from methyl salicylate, 1-naphthol, 7-hydroxy-2-methylbenzo[b]furan, and 8-hydroxycoumarin showed the comparable activity to that of DDI against HIV-1.     

Generation of 6-(Trifluoromethyl)-4,5-dihydro-2(3H)-pyridone and the Application to Synthesis of Some Fused Nitrogen Heterocycles Carrying a Trifluoromethyl Group on the Bridgehead Position via Radical Cyclization of Dihydropyridones

Staudinger/aza-Wittig reaction of 6,6,6-trifluoro-5-oxohexanoyl azide with PPh(3) or PBu(3) was examined. A reactive intermediate acyl imine 1 was trapped by methanol. Without nucleophile, isomerized enamide form 3 was obtained. N-Iodobenzoylation and N-haloalkylation of 3 and following radical cyclization via the 5-exo or 6-exo mode gave benzoindolizidinone, indolizidinone, and quinolizidinone derivatives 10-14, which have a trifluoromethyl group at the bridgehead position adjacent to nitrogen. Although LiAlH(4) reduction of 10 and 11 gave a mixture of saturated benzoindolizidine 15 and amino alcohol 16, reduction with BH(3)/THF selectively gave the desired 15 and indolizidine 17 from 13.     

High throughput continuous titration based on a flow ratiometry controlled with feedback-based variable triangular waves and subsequent fixed triangular waves

We propose a new approach for high-throughput continuous titration based on a flow ratiometry. The method was applied to potentiometric titrations of acids and bases. A base solution, the flow rate F_B of which was varied in response to controller output voltage V_c, was merged with an acid solution under constant total (acid + base) flow rate. Downstream, the pH of the mixed solution was measured with a flow-through glass electrode. Initially, V_c, and thus F_B was increased linearly. At the instant the equivalence point (EP) was sensed, the ramp direction of V_c was reversed from upward to downward. The direction was reversed to upward when EP was sensed again. Such the feedback-based operation gives a triangular waveform of V_c, because there is a delay corresponding mainly to the transit time of merged solutions to reach the sensor. The value of V_c that gives EP composition, V_E, was estimated by averaging the most recent maximum and minimum values of V_c. Next, fixed triangular waves of V_c was used to control F_B. The amplitude and the scan rate of the waves were fixed narrower and faster, respectively, than those in the feedback-based operation in order to improve the throughput rate. The EP can be located as long as the scan range covers V_E. These automated processes limited the titration to just the narrow range around EP, and thus realized extremely high throughput rate of maximally 17.6 titrations per minute (=3.4 s per titration) at R.S.D. = 0.35%.     

Total synthesis and absolute stereochemistry of pentenocin B, a novel interleukin-1 beta converting enzyme inhibitor

Four possible diastereomers of pentenocin B were synthesized in a stereocontrolled manner, and the first total synthesis of a natural enantiomer of (+)-pentenocin B unequivocally established the absolute stereochemistry to be 4S,5R,6R.