Synthesis and Study of Copper-Containing Polymers of Microcrystalline Cellulose Sulfates from Larch Wood

For the first time, the synthesis of water-soluble copper-containing microcrystalline cellulose sulfates (Сu-MCS) has been performed by the ion exchange method. The composition of the products has been studied by chemical methods and X-ray spectral microanalysis. The copper content in the Сu-MCS samples was 12.6–14.1%. The absence of sodium in the resulting polymer indicates the complete substitution of the sodium cations by the copper cations in the sodium salt of MCC sulfate. The structure of the copper-containing sulfates of microcrystalline cellulose has been confirmed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and electron paramagnetic resonance (EPR). According to the XRD method, Сu-MCS and Na-MCS have an amorphous structure in contrast to the original MCC samples, which have a high degree of crystallinity. The EPR data have demonstrated the formation of a pseudocrystalline structure of the copper-containing salt system in the Сu-MCS samples. As shown by atomic-force microscopy, the surface of the Сu-MCS films consists of homogeneous crystallites, which have a spherical or slightly extended form with the size of about 70 nm. The film surface is quite homogeneous in its phase composition and contains no impurities.

     

Humic Acids as Electron Acceptors for Anaerobic Microbial Oxidation of Vinyl Chloride and Dichloroethene

Anaerobic oxidation of [1,2-¹⁴C]vinyl chloride and [1,2-¹⁴C]dichloroethene to ¹⁴CO₂ under humic acid-reducing conditions was demonstrated. The results indicate that waterborne contaminants can be oxidized by using humic acid compounds as electron acceptors and suggest that natural aquatic systems have a much larger capacity for contaminant oxidation than previously thought.     

Synthesis of Photolabile Caged Amino Acids for Measuring Amino Acid Transporters

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.     

Preparation of Two Series of Oligo-guluronic Acids from Sodium Alginate by Acid Hydrolysis and Enzymatic Degradation

The objective of this study was to prepare two series of authentic oligo-guluronic acids from sodium alginate. Oligo-guluronic acids (DP = 1–9) were prepared from an acid hydrolysate of poly-guluronic acid by successive chromatographies of Bio-Gel P-6 and Q Sepharose Fast Flow. Oligo-guluronic acids having 4-deoxy-l-erythro-hex-4-enopyranosyluronic acid residues at the non-reducing end (DP = 2–7) were prepared from the enzymatic degradation products of the poly-guluronic acid in the same manner. Each of the isolated oligo-guluronic acids gave a single band on fluorophore-assisted carbohydrate electrophoresis. These results suggest that successive chromatographies used in this study are well suited for the preparation of alginate-derived oligouronic acids.     

The Effect of Microcrystalline Cellulose Crystallinity on the Hydrophilic Property of Tablets and the Hydrolysis of Acetylsalicylic Acid as Active Pharmaceutical Ingredient Inside Tablets

The crystal structures of active pharmaceutical ingredients and excipients should be strictly controlled because they influence pharmaceutical properties of products which cause the change in the quality or the bioavailability of the products. In this study, we investigated the effects of microcrystalline cellulose (MCC) crystallinity on the hydrophilic properties of tablets and the hydrolysis of active pharmaceutical ingredient, acetylsalicylic acid (ASA), inside tablets by using tablets containing 20% MCC as an excipient. Different levels of grinding were applied to MCC prior to tablet formulation, to intentionally cause structural variation in the MCC. The water penetration and moisture absorbability of the tablets increased with decreasing the crystallinity of MCC through higher level of grinding. More importantly, the hydrolysis of ASA inside tablets was also accelerated. These results indicate that the crystallinity of MCC has crucial effects on the pharmaceutical properties of tablets even when the tablets contain a relatively small amount of MCC. Therefore, controlling the crystal structure of excipients is important for controlling product qualities.     

Synthesis and Antiviral Activity of Betulonic Acid Amides and Conjugates with Amino Acids

Betulonic acid amides with aliphatic and heterocyclic amines and with L-amino acids were synthesized by the acid chloride method. Betulonic acid amide and L-methionine derivatives of betulonic acid and its 3-oxime effectively inhibit the influenza A virus. Betulonic acid octadecylamide is active against the herpes simplex Type 1 virus. The conjugate of betulonic acid 3-oxime with L-methionine is also active toward HIV-1. The tested compounds mainly show no activity toward the ECHO6 virus, which is devoid of a coat.     

Estimations of Uronic Acids as Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples

The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrated in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideuterated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater.     

Ethyl Cellulose Microcapsules for Protecting and Controlled Release of Folic Acid

Ethyl cellulose microcapsules were developed for use as a drug-delivery device for protecting folic acid from release and degradation in the undesirable environmental conditions of the stomach, whilst allowing its release in the intestinal tract to make it available for absorption. The controlled release folic acid-loaded ethyl cellulose microcapsules were prepared by oil-in-oil emulsion solvent evaporation using a mixed solvent system, consisting of a 9:1 (v/v) ratio of acetone:methanol and light liquid paraffin as the dispersed and continuous phase. Span 80 was used as the surfactant to stabilize the emulsion. Scanning electron microscopy revealed that the microcapsules had a spherical shape. However, the particulate properties and in vitro release profile depended on the concentrations of the ethyl cellulose, Span 80 emulsifier, sucrose (pore inducer), and folic acid. The average diameter of the microcapsules increased from 300 to 448 μm, whilst the folic acid release rate decreased from 52% to 40%, as the ethyl cellulose concentration was increased from 2.5% to 7.5% (w/v). Increasing the Span 80 concentration from 1% to 4% (v/v) decreased the average diameter of microcapsules from 300 to 141 μm and increased the folic acid release rate from 52% to 79%. The addition of 2.5–7.5% (w/v) of sucrose improved the folic acid release from the microcapsules. The entrapment efficiency was improved from 64% to 88% when the initial folic acid concentration was increased from 1 to 3 mg/ml.     

Solid Phase Synthesis of a Hydroxypyrrolidine Derivative and its Use in Solid Phase Peptide Synthesis as Constrained Statine Mimic

As part of our efforts to design constrained peptide mimics and introduce them in peptide sequences, we set up the synthesis of racemic N-Fmoc protected hydroxypyrrolidine by reduction of the corresponding oxopyrroline. Hydroxypyrrolidines are synthesized using amino acid building block and β-ketoester via a 4-steps solid supported route on Wang resin beads. The hydroxypyrrolidine template can be seen as a constrained mimic of statine. As proof of concept, the pseudopeptide JMV 2776, incorporating this new statine mimic has been synthesized. We replaced the phenyl statine building block in the sequence of known BACE 1/2 inhibitors by 5-benzyl 2-methyl 4-hydroxypyrrolidine, using conventional Fmoc SPPS on Rink amide PS resin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Solid Phase and Solution Phase Synthesis of Gamma Amino Acid Homo-Oligomers and Mixed Oligomers

Abstract  

An efficient stepwise synthesis of homo-oligomers and mixed oligomers of gabapentin and pregabalin on solid support using Fmoc-protected derivatives and HBTU/HOBt/DIEA as coupling agent is described. The synthesis was also carried out using solution phase methodology. The Gpn/Pgn homo oligomers and mixed oligomers forms C₉ helix in solution as determined by NMR study. Chiral as well as achiral gamma amino acids were used for the synthesis of oligomers in order to investigate the secondary structural preferences.     

葡萄糖基聚合物的酶催化合成及其在制备缓释微球中的应用

以脂肪酶Novozym-435催化葡萄糖和10-十一碳烯酸合成了6-O-(10-十一碳烯酸)-葡萄糖酯,在K2S2O8引发下合成了葡萄糖基聚合物.采用复凝聚法以葡萄糖基聚合物和海藻酸钠为基质材料,将非诺洛芬钙包裹制成缓释微球.通过L9(34)正交实验得出微球的最佳制备工艺条件,结果是葡萄糖基聚合物:海藻酸钠质量比=1:2,pH 3.0,搅拌速度400 r/min,反应成球温度45℃.在最佳工艺条件下制备的非诺洛芬钙缓释微球,粒径范围是10～20μm,平均药物包封率(73.74±3.12)%.同时,体外溶出试验表明,该微球具有较好的缓释作用.     

The Effects of Borate Minerals on the Synthesis of Nucleic Acid Bases,Amino Acids and Biogenic Carboxylic Acids from Formamide

The thermal condensation of formamide in the presence of mineral borates is reported. The products afforded are precursors of nucleic acids, amino acids derivatives and carboxylic acids. The efficiency and the selectivity of the reaction was studied in relation to the elemental composition of the 18 minerals analyzed. The possibility of synthesizing at the same time building blocks of both genetic and metabolic apparatuses, along with the production of amino acids, highlights the interest of the formamide/borate system in prebiotic chemistry.     

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

Regulation of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The regulation of synthesis of valyl-, leucyl-, and isoleucyl-transfer ribonucleic acid (tRNA) synthetases was examined in strains of Escherichia coli and Salmonella typhimurium. When valine and isoleucine were limiting growth, the rate of formation of valyl-tRNA synthetase was derepressed about sixfold; addition of these amino acids caused repression of synthesis of this enzyme. The rate of synthesis of the isoleucyl- and leucyl-tRNA synthetases was derepressed only during growth restriction by the cognate amino acid. Restoration of the respective amino acid to these derepressed cultures caused repression of synthesis of the aminoacyl-tRNA synthetase, despite the resumption of the wild-type growth rate.     

Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production

We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.Production of bioethanol from biomass has recently attracted attention due to the mandate for a billion gallons of renewable fuel by the new Energy Policy Act (22). Current production processes using sugar cane and cornstarch are well established (19, 23). However, utilization of a cheaper substrate would render bioethanol more competitive with fossil fuel (29). Cellulosic biomass found in many low-value agricultural or wood pulping wastes is particularly well suited because of its large-scale availability, low cost, and environmentally benign production (23). The primary obstacle impeding the more widespread production of ethanol from cellulose is the absence of a low-cost technology for overcoming its recalcitrant nature (21).Recently, a new method known as consolidated bioprocessing (CBP) has been proposed that combines enzyme production, cellulose saccharification, and fermentation into a single process to dramatically reduce the cost of ethanol production (22). An ideal microorganism for CBP should possess the capability of simultaneous cellulose saccharification and ethanol fermentation. One attractive candidate is Saccharomyces cerevisiae, which is widely used for industrial ethanol production due to its high ethanol productivity and high inherent ethanol tolerance (24). Attempts have been made to engineer S. cerevisiae to hydrolyze cellulose (6, 7, 16). However, due to energetic limitations under anaerobic conditions, only a small amount of cellulases can often be secreted. An alternative is to display the cellulolytic enzymes on the yeast cell surface (13, 14). Up to three different cellulases have been displayed, permitting the hydrolysis of cellulose with concomitant ethanol production. While these results point to a potential strategy of combining ethanol-producing capability with cellulose hydrolysis, the efficiency of hydrolysis must be significantly improved before it can be employed for practical applications.Many anaerobic bacteria have developed an elaborately structured enzyme complex on the cell surface, called the cellulosome, to maximize the catalytic efficiency of cellulose hydrolysis with only a limited amount of enzymes (1, 8, 9). The major component of these cellulosome complexes is a structural scaffoldin consisting of at least one cellulose-binding domain (CBD) and repeating cohesin domains, which are docked individually with a different cellulase tagged with the corresponding dockerin domain (26). Since the interaction between dockerin and cohesin is species specific (17, 25), designer minicellulosomes composed of three different dockerin-cohesin pairs with a cellulose hydrolysis efficiency up to sixfold higher than that of similar free enzymes have been generated (11, 12). Recently, it has been shown that the specific cellulose hydrolysis rates of metabolically active cultures of C. thermocellum displaying cellulosomes are more than fourfold higher than those of purified cellulosomes (20). This significant improvement appears to be a surface phenomenon involving adhesion to cellulose for enhanced substrate capture.In the present report, we demonstrate the functional assembly of a minicellulosome composed of three different cellulases on the S. cerevisiae cell surface and the feasibility of using the engineered yeast strains for cellulosic ethanol production. The success of displaying a functional cellulosome on the surface of an organism that already produces high titers of ethanol could lay a foundation for the achievement of an industrially relevant CBP-enabling microorganism.     

Polyethyleneglycol-Based Resins as Solid Supports for the Synthesis of Difficult or Long Peptides

An evaluation of the polyethyleneglycol-based ChemMatrix^? resin as solid support for the synthesis of challenging peptide sequences is presented. Comparison with conventional polystyrene and polyethyleneglycol-polystyrene resins in several instances of typically difficult solid phase syntheses shows a consistently better performance of the ChemMatrix^? resin in terms of end product purity. Representative test sequences include a 15-residue antibiotic, a gp41 ectodomain hybrid sequence, a calcipressin fragment with an N-terminal Arg₁₁ extension, and two chemokines of 69- and 64-amino acid residues. Interestingly, a difference in only five amino-acids between the two chemokine sequences had a remarkable impact on synthetic results, which in the case of the 69-residue peptide required additional refinements (β-sheet-breaking pseudoproline dipeptides) for success. This paper is dedicated to the memory of Bruce Merrifield, a dear teacher, mentor and friend.     

辐射引发合成高吸水树脂及用于吊兰栽培研究

高吸水树脂是一种能吸收并保持大量植物所需水分和营养成分的,并可重复利用的高分子吸水材料。本实验通过60Coγ射线辐射诱导丙烯酰胺,得到一种新型耐降解吸水树脂。为研究新型吸水树脂对园艺作物吊兰的生长及氮素养分吸收的影响,探索其作为栽培基质-水晶泥的应用前景。本研究采用室内盆栽模拟试验,通过15N同位素示踪技术研究了大（0.75 cm）、中（0.5 cm）、小（0.25 cm）三种粒径水晶泥基质对园艺作物吊兰体内氮素分配及其养分利用效率的影响。结果表明：小粒径水晶泥基质栽培下,叶部的吸氮量占比最大,达到52.169%,增加其观赏性;其次,小粒径水晶泥基质栽培下,吊兰对氮素养分的利用效率最高,达到了32.397%,但与其他两处理间的差异均未达到显著水平。     

Amino Acid Nucleic Acids: Synthesis and Hybridization Properties of a Novel Class of Antisense Oligonucleotides

Abstract

Oligonucleotides containing novel phoshoramidite 12 were synthesized and studied for their hybridization properties for the first time. Interestingly, these modified oligonucleotides showed a remarkable resistance to exonuclease.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.