Synthesis of Optically Active Cyanohydrins Using R-Oxynitrilase in a Liquid-Liquid Biphasic System: Part 1: An Industrially useful Procedure

An improved method is presented for preparing optically active cyanohydrins in high yield and high enantioselectivity, using R-oxynitrilase from almonds as the catalyst in a biphasic water-organic solvent system. Reaction conditions for two representative substates have been studied with respect to conversion, optical purity and enzyme stability.

The suitability of the procedure for industrial use was substantiated by the possibility of reusing the enzyme-containing aqueous phase for the synthesis of R-mandelonitrile.     

Adsorptive control of water in esterification with immobilized enzymes: II. fixed-bed reactor behavior

Experimental and theoretical studies are conducted to understand the dynamic behavior of a continuous-flow fixed-bed reactor in which an esterification is catalyzed by an immobilized enzyme in an organic solvent medium. The experimental system consists of a commercial immobilized lipase preparation known as Lipozyme as the biocatalyst, with propionic acid and isoamyl alcohol (dissolved in hexane) as the reaction substrates. A complex dynamic behavior is observed experimentally as a result of the simultaneous occurrence of reaction and adsorption phenomena. Both propionic acid and water are adsorbed by the biocatalyst resulting in lower reaction rates. In addition, an excessive accumulation of water in the reactor leads to a rapid irreversible inactivation of the enzyme. A model based on previously-obtained adsorption isotherms and kinetic expressions, as well as on adsorption rate measurements obtained in this work, is used to predict the concentration and thermodynamic activity of water along the reactor length. The model successfully predicts the dynamic behavior of the reactor and shows that a maximum thermodynamic activity of water occurs at a point at some distance from the reactor entrance. A cation exchange resin in sodium form, packed in the reactor as a selective water adsorbent together with the catalyst particles, is shown to be an effective means for preventing an excessive accumulation of water formed in the reaction. Its use results in longer cycle times and greater productivity. As predicted by the model, the experimental results show that the water adsorbed on the catalyst and on the ion exchange resin can be removed with isoamyl alcohol with no apparent loss in enzyme activity.     

Synthesis of Optically Active Cyanohydrins Using R-Oxynitrilase in a Liquid-Liquid Biphasic System: Part 1: An Industrially useful Procedure

An improved method is presented for preparing optically active cyanohydrins in high yield and high enantioselectivity, using R-oxynitrilase from almonds as the catalyst in a biphasic water-organic solvent system. Reaction conditions for two representative substates have been studied with respect to conversion, optical purity and enzyme stability.

The suitability of the procedure for industrial use was substantiated by the possibility of reusing the enzyme-containing aqueous phase for the synthesis of R-mandelonitrile.     

Trifluoroacetic anhydride-catalyzed nitration of toluene as an approach to the specific analysis of nitrate by gas chromatography-mass spectrometry.

The nitration of aromatic compounds by electrophilic substitution is often utilized in analyses of nitrate concentrations in physiological samples by gas chromatographic methods. Problems associated with the use of concentrated sulfuric acid, which is normally used to catalyze this reaction, led us to investigate an alternative method. We describe here a facile GC/MS assay for nitrate in plasma or urine samples which takes advantage of the ability of trifluoroacetic anhydride (TFAA) to catalyze the nitration of aromatics. Toluene, utilized as both reaction solvent and electrophile, was shown to react with nitrate in the presence of TFAA to quantitatively produce the three nitrotoluene isomers (ratio o-:m:p-, approx 57:3:40). Following the incorporation of 15N-labeled nitrate as internal standard, nitrotoluene was quantified using GC/MS by analysis of the selected the ion pairs m/z 120 and 121 (M+ -OH) for the o-isomer or m/z 137 and 138 (molecular ion, M+) for the p-isomer. The limit of detection for nitrate after TFAA-catalyzed conversion to nitrotoluene was less than 100 fmol on column (s/n; 40:1). The TFAA-based GC/MS assay was compared with that utilizing the usual catalyst, concentrated sulfuric acid. With the exception of samples containing nitroarginine analogues, good correlation was found for urine or plasma samples analyzed using either a standard sulfuric acid-catalyzed method or the TFAA-catalyzed procedure. Nitroarginine analogues, which can be present in samples following their use as nitric oxide synthase inhibitors, did not decompose under the conditions of the TFAA-catalyzed assay and, hence, do not give rise to significant interference with nitrate analysis in this procedure. In contrast, catalytic sulfuric acid caused nitroarginine analogues to decompose (essentially quantitatively) and cause spuriously high nitrate levels in samples. The use of TFAA as a catalyst for the nitration of toluene enables a facile and sensitive GC/MS analysis for nitrate which offers improved safety and sample integrity.     

Low reaction temperature increases the selectivity in an enzymatic reaction due to substrate solvation effects

Immobilized a-chymotrypsin was used as catalyst for studying temperature effects on acyl transfer reactions (acyl-donor: Bz-TyrOEt) in a water-immiscible organic solvent. The solubility of the two nucleophiles, Phe-NH and water, decreased with decreasing temperature. The relative decrease for the amide was larger (2.4-fold) than for water. Therefore the thermodynamic activity (estimated by the relative saturation) increased more for this substrate and hence the selectivity in the reaction was increased.     

An integrated process: ester synthesis in an enzymatic membrane reactor and water sorption

In the case of such reactions as ester synthesis, water is produced during the reaction. Because these reactions are carried out in hydrophobic solvents an additional (water) phase in the system must not be allowed, i.e. the concentration of water saturation in the organic solvent should not be exceeded. In such a case, the reaction kinetics and product equilibrium concentration undergo undesirable changes because of the partition coefficient of the components and hampered process of product separation. Hence, removal of the water produced in the reaction determines whether the process is successful or not. For this purpose, the integrated process with water sorption in the column with molecular sieves was applied. Integration of the process of synthesis and dehydration of a reaction phase, in which a biocatalyst is suspended and not dissolved as in water solutions, requires holding up of the catalyst in the reactor before directing the stream of reaction mixture to dehydration process. This hold-up and a possibility of multiple use of the catalyst may be accomplished by using a separating barrier, e.g. an ultrafiltration membrane or by permanent fixing of the catalyst to the matrix, e.g. a polymeric membrane. The efficiency and activity of a biocatalyst (lipase CAL-B) immobilized on a polymer membrane by sorption and chemical binding, were determined. A subject of study was the synthesis of geranyl acetate, one of the most known aromatic compound. A hydrophobic (polypropylene) matrix was shown to be a much better carrier in the reactions performed in an organic solvent than a hydrophilic (polyamide) membrane being tested. The reaction kinetics of geranyl acetate synthesis with the use of geraniol and acetic acid as substrates, was described by the equation defining the "Ping-Pong Bi Bi" mechanism that was related additionally to the inhibition of a substrate (acetic acid). The following constants of kinetic equation were obtained k(3)(')=0.344 mol g(-1)h(-1), K(mA)=0.257 mol l(-1), K(mG)=1.629 and K(iA)=0.288 for the native enzyme and v(max,Gel)=111.579 mol l(-1)h(-1), K(mA)=0.255 mol l(-1), K(mG)=1.91 mol l(-1), K(iA)=0.238 mol l(-1) for the one immobilized by sorption on a polypropylene membrane. Half-life time of the native enzyme activity was 204 h and stability of the immobilized preparation was 70 h. With respect to the reaction kinetics and stability of the native enzyme and immobilized preparation, from both types of membrane bioreactor more attractive appears to be the one in which the membrane is used not as a catalyst layer but only as a barrier that immobilizes the native enzyme within the bioreactor volume. When an integrated process proceeds, the method to collect water in the sorption column during the process, appeared to work very well. The reaction proceeded with a very high efficiency (after 120 h alpha=98.2% for native enzyme and 83.2% for immobilized enzyme) and due to low water concentration in the system ( approximately 0.000% v/v) the second phase was not created.     

Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications

A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent. (c) 1994 John Wiley & Sons, Inc.     

Solvent effects on biocatalysis in organic systems: equilibrium position and rates of lipase catalyzed esterification

Porcine pancreatic lipase immobilized on celite particles has been employed as a catalyst for the esterification of dodecanol and decanoic acid in a predominantly organic system. Solvent influence on the equilibrium position and on the catalyst activity has been studied using 20 solvents, including aliphatic and aromatic hydrocarbons, ethers, ketones, nitro- and halogenated hydrocarbons, and esters. The equilibrium constant for esterification correlates well with the solubility of water in the organic solvent, which in turn shows a good relationship with a function of Guttman's donor number and the electron pair acceptance index number of the solvent. This may be rationalized in terms of the requirements for solvation of water and of the reactants. The catalyst activity, measured as the initial rate of the esterification reaction, is best correlated as a function of both n-octanol-water partition coefficient (log P) and either the electron pair acceptance index or the polarizability.     

微波辐射法合成苄基-2-萘基醚的研究

在微波辐射条件下,以2-萘酚和氯化苄为原料,用氢氧化钠作碱剂,碘化钾作催化剂,以水和N,N-二甲基甲酰胺(DMF)为溶剂合成了苄基-2-萘基醚;采用单因素实验法,考察了反应物的摩尔比、催化剂用量、微波功率、辐射时间等因素对苄基-2-萘基醚产率的影响。实验结果表明:在n(2-萘酚):n(氢氧化钠):n(氯化苄):n(碘化钾)=1:1.1:1:0.018,水7 ml,DMF 25 ml,微波功率320 W和辐射时间75 s的优化条件下,苄基-2-萘基醚的产率可达88.03%。     

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10^-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.     

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.     

Antioxidant capacity and environmentally friendly synthesis of dihydropyrimidin-(2H)-ones promoted by naturally occurring organic acids

The Biginelli reaction is a multicomponent reaction involving the condensation between an aldehyde, a β-ketoester, and urea or thiourea, in the presence of an acid catalyst, producing dihydropyrimidinones (DHPMs). Owing to their important pharmacological properties, the DHPMs have been studied by many authors. However, most of the methodologies used for the synthesis of these compounds require drastic reaction conditions. In the current study, we report an efficient and clean procedure for preparing DHPMs by the use of citric acid or tartaric acid as a promoter of the Biginelli synthesis in ethanol as solvent. In addition, we have evaluated the antioxidant capacity of the compounds synthesized by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and the thiobarbituric acid-reactive species test. Two compounds presented antioxidant activity and also reduced lipid peroxidation at concentrations of 200 and 300 μM. In summary, we report an environmentally friendly procedure for the preparation of DHPMs and demonstrate the antioxidant capacity of some of the compounds.     

The physical origin of the low solubility of nonpolar solutes in water

Elementary but general statistical-mechanical relations are derived that relate the thermodynamic properties of the dissolution process to those of the pure solvent. A number of conclusions are drawn from qualitative arguments that these relations suggest. These include the following: (1) The low solubility of nonpolar solutes in water arises not from the fact that water molecules can form hydrogen bonds, but rather from the fact that they are small in size. (2) The large entropy decrease attending the transfer of an inert solute from a nonaqueous solvent to water is largely due to the decrease in entropy of the nonaqueous solvent as the solvent–solvent interaction is restored on removal of the solute from it. (3) It is improper to use values of thermodynamic quantities obtained from small-molecule transfer studies for those that involve macromolecular folding and interaction.     

Concerted and stepwise dehydration mechanisms observed in wild-type and mutated Escherichia coli dTDP-glucose 4,6-dehydratase

The conversion of dTDP-glucose into dTDP-4-keto-6-deoxyglucose by Escherichia coli dTDP-glucose 4,6-dehydratase (4,6-dehydratase) takes place in the active site in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene, and rereduction of C6 to the methyl group. The 4,6-dehydratase makes use of tightly bound NAD(+) as the coenzyme for transiently oxidizing the substrate, activating it for the dehydration step. Dehydration may occur by either of two mechanisms, enolization of the dTDP-4-ketoglucose intermediate, followed by elimination [as proposed for beta-eliminations by Gerlt, J. A., and Gassman, P. G. (1992) J. Am. Chem. Soc. 114, 5928-5934], or a concerted 5,6-elimination of water from the intermediate. To assign one of these two mechanisms, a simultaneous kinetic characterization of glucosyl C5((1)H/(2)H) solvent hydrogen and C6((16)OH/(18)OH) solvent oxygen exchange was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The reaction of the wild-type enzyme is shown to proceed through a concerted dehydration mechanism. Interestingly, mutation of Asp135, the acid catalyst, to Asn or Ala alters the mechanism, allowing enolization to occur to varying extents. While aspartic acid 135 is the acid catalyst for dehydration in the wild-type enzyme, the differential enolization capabilities of D135N and D135A dehydratases suggest an additional role for this residue. We postulate that the switch from a concerted to stepwise dehydration mechanism observed in the aspartic acid variants is due to the loss of control over the glucosyl C5-C6 bond rotation in the active site.     

A facile and effective synthesis of dinucleotide 5'-triphosphates

We report on the successful synthetic procedure for the conversion of 5'-monophosphorylated 2'-deoxydinucleotides into their 5'-triphosphate derivatives in satisfactory to excellent yields. The activation of the terminal phosphate group was achieved under the Mukaiyama conditions in the presence of a nucleophilic catalyst. The reaction conditions (solvent, counter ions, activation time and reagent excess) were optimized for all dinucleotides.     

Automated solid-phase synthesis of metabolically stabilized triazolo-peptidomimetics

The use of 1,4-disubstituted 1,2,3-triazoles as trans-amide bond surrogates has become an important tool for the synthesis of metabolically stabilized peptidomimetics. These heterocyclic bioisosters are generally incorporated into the peptide backbone by applying a diazo-transfer reaction followed by CuAAC (click chemistry) with an α-amino alkyne. Even though the manual synthesis of backbone-modified triazolo-peptidomimetics has been reported by us and others, no procedure has yet been described for an automated synthesis using peptide synthesizers. In order to efficiently adapt these reactions to an automated setup, different conditions were explored, putting special emphasis on the required long-term stability of both the diazo-transfer reagent and the Cu(I) catalyst in solution. ISA·HCl is the reagent of choice to accomplish the diazo-transfer reaction; however, it was found instable in DMF, the most commonly used solvent for SPPS. Thus, an aqueous solution of ISA·HCl was used to prevent its degradation over time, and the composition in the final diazo-transfer reaction was adjusted to preserve suitable swelling conditions of the resins applied. The CuAAC reaction was performed without difficulties using [Cu (CH₃CN)₄]PF₆ as a catalyst and TBTA as a stabilizer to prevent oxidation to Cu(II). The optimized automated two-step procedure was applied to the synthesis of structurally diverse triazolo-peptidomimetics to demonstrate the versatility of the developed methodology. Under the optimized conditions, five triazolo-peptidomimetics (8–5 amino acid residues) were synthesized efficiently using two different resins. Analysis of the crude products by HPLC-MS revealed moderate to good purities of the desired triazolo-peptidomimetics (70–85%). The synthesis time ranged between 9 and 12.5 h.     

Chemiluminescence of iron-chlorophyllin.

The iron-chlorophyllin complex was found to be chemiluminescent (CL) in an acetonitrile (22%)/water mixed solvent. In the presence of hydrogen peroxide, when iron-chlorophyllin was added to the mixed solvent, a sharp CL signal immediately appeared. Also, analysis of the absorption spectra revealed decomposition of iron-chlorophyllin (based on decrease in absorbance at 396 nm), hence iron-chlorophyllin is the CL substance. Moreover, the CL intensity decreased in the presence of potassium thiocyanate (KSCN), indicating that the axial coordinative position of iron-chlorophyllin acts as a point of catalytic activation. In addition, when fluorophores were present with iron-chlorophyllin CL, their CL intensity values were similar to or greater than that of the well-known trichlorophenylperoxalate ester (TCPO) CL. Thus, during the decomposition reaction of iron-chlorophyllin, the latter transfers its energy to the coexisting fluorophores. Moreover, since the decomposed compound in this CL reaction had a fluorescence, it was found that the iron-chlorophyllin also functions as an energy donor. Therefore, the iron-chlorophyllin complex acts not only as a CL substance, but also as a catalyst and energy donor in the reaction.     

Polychrome Staining of Epoxy Semithin Sections Using Cacodylic Buffer as a Stain Solvent

Many polychromatic stains are in use for epoxy-embedded tissues (Horobin 1983). We should like to report a quick and easy polychromatic staining procedure that we find useful for routine use. Formerly the stain we used was prepared in 20 ml water and 5 ml 96% alcohol, and gave polychromatic staining only at pH 7.4 obtained by the addition of 1 N NaOH. However, the stain gave satisfactory results only for two or three days. We found that stabilization of the staining solution through the use of an ethyl alcohol-cacodylic buffer solvent increased the life of the staining solution. This was convenient because the cacodylic buffer is used in our laboratory as a component of fixatives, and is not prepared specially for the staining.     

Highly active, recyclable catalyst for the manufacture of viscous, low molecular weight, CO-ethene-propene-based polyketone, base component for a new class of resins

A highly active, recyclable homogeneous palladium(II) catalyst is described for the manufacture of viscous, low molecular weight CO-ethene-propene-based polyketone (CariliteOligomer), used for the manufacture of a new class of resins (CariliteResins). The catalyst is composed of palladium acetate, and a sulfonated diphosphine ligand, bdompp-S (1,3-bis(di-(2-methoxy, 5-sulfonatophenyl)phosphino)propane). In comparison with its non-sulfonated counterpart this catalyst not only exhibits a much more favourable partitioning coefficient in liquid-liquid separation of the polyketone product and solvent, but it also exhibits an approximately 2.5 times higher catalytic activity (up to 11.2 kg PK (g Pd)⁻¹ h⁻¹) in the manufacture of PK-PE-30 (polyketone terpolymer built up of CO, ethene and propene in a molar ratio of 100:30:70). A variety of salts were found to exert a positive influence on the activity of the catalyst. Possible origins of this ‘salt-effect’ are briefly discussed. The bdompp-S ligand was synthesised by sulfonation of bdompp using either a boric acid-oleum mixture or sulfuric acid as the sulfonation reagent. The product was isolated either as sodium-salt (bdompp-S[Na]₄·nNa₂SO₄), by extraction with methanol after neutralisation, or, in acidic, hydrated form (bdompp-S[H]₄·nH₂O), via a new and highly efficient procedure, i.e. cooling the reaction mixture after dilution with water. The X-ray crystal structure of bdompp-S[H]₄·nH₂O is discussed.