Multicomponent reactions in crop protection chemistry

An overview is given of the significance of multicomponent reactions in the synthesis of agrochemicals. The most important applications of multicomponent condensations, such as the Biginelli reaction, Bucherer-Bergs reaction, Hantzsch dihydropyridine synthesis, Kabachnik-Fields reaction, Mannich reaction, Passerini reaction, Petasis reaction, Strecker reaction, Ugi reaction and Willgerodt-Kindler reaction, to the synthesis of herbicidally, fungicidally and insecticidally active compounds are presented. Also the mode of action and biological activity of these multicomponent reaction products are reported.     

Enzymatic hydrolysis of hydrophobic triolein by lipase in a mono-phase reaction system containing cyclodextrin; Reaction characteristics

A hydrophobic substrate triolein was hydrolyzed by lipase in a mono-phase reaction system containing cyclodextrin(CD) as emulsifier. The triolein was transformed to an emulsion-like state in the CD containing reaction system in contrast to the oil-droplet like state without CD due to the formation of an inclusion complex between the lipids and CDs. The hydrolysis reaction increased substantially in the CD containing reaction system, and the optimum reaction conditions including the amount of lipase, β-CD concentration, and mixing ratio of triolein and β-CD, were determined. The performance of the enzyme reaction in a mono-phase reaction system was compared with that of a two-phase reaction system which used water immiscible hexane as the organic solvent. The role of a CD in the mono-phase reaction system was elucidated by comparing the degree of the inclusion complex formation with triolein and oleic acid, K_m and V_max values, and product inhibition by oleic acid in aqueous and CD containing reaction systems. The resulting enhanced reaction seems to be caused by two phenomena; the increased accessibility of lipase to triolein and reduced product inhibition by oleic acid through the formation of an inclusion complex.     

Reaction rate calculations for cosubstrates diffusing into catalyst layer from opposite sides

The effects of diffusion on a reaction taking place in a permeable catalyst are examined theoretically for the case where the reaction has two substrates supplied from opposite sides of a catalytic slab. The solutions of the reaction-diffusion equation for combinations of zeroth-and first-order kinetics are given in terms of an effectiveness factor and a parameter describing the position in the layer where the reaction occurs. In these terms, the results vary only weakly with reaction order. The use of the exact solutions for a reaction that is zeroth order in both sub strates is proposed as a general rule for estimating the reaction rate and the reaction position.     

A comparative study on the thermal decomposition of four cereal straws in an oxidizing atmosphere

The thermal decomposition characteristics of four types (wheat, barley, oats and rye) of cereal straws were studied. Two varieties from each type of straw were used. The thermal degradation behaviours and kinetic parameters (order of reaction, activation energy and preexponential factor) of the straws were compared. Two distinct reaction zones were observed for all types and varieties of straws. Thermal degradation rates in the first reaction zone were significantly higher than those in the second reaction zone. The activation energy was in the ranges of 80–102 kJ/mol and 34–75 kJ/mol, whereas the order of reaction was in the ranges of 1·3–2·3 and 0·1–0·7 for the first and second reaction zones, respectively. The Shaw variety of oats straw had the highest activation energies (102 and 75 kJ/mol) and reaction orders (2·3 and 0·7) in both the first and second reaction zones, respectively. The lowest activation energy (80 kJ/mol) and order of reaction (1·3), in the first reaction zone, corresponded to Absolvant and Monopol wheat straws. The activation energies and reaction orders of barley and rye straws were in the ranges of 85–94 kJ/mol and 1·9–2·3, respectively. There was not any significant difference between the rate constants of the straw varieties, in the first reaction zone. However, oats straws had significantly higher rate constants in the second reaction zone as compared to the rate constants of wheat, barley and rye straws.     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

Free radicals in the flavin reduction reaction. Interaction of reduced nicotinamide adenine dinucleotide with flavin in solution]

The study of the flavin mononucleotide (FMN)-reduced nicotinamide adeninedinucleotide (NADH) reaction was carried out both under aerobic and anaerobic conditions, using spectrophotometric and titrimetric methods. The consumption of NADH was shown to exceed two times the consumption of FMN in the anaerobic reaction and the rate constant in the aerobic reaction was found to be about 4 times of that of the anaerobic reaction. Moreover, the replacement of anaerobic conditions by aerobic ones at pH 5.0 resulted in a considerable increase of proton consumption rate in the reaction course. The data obtained are contradictory to the generally accepted hypothesis of hydrid-ion transfer in the reaction of NADH oxidation. It was assumed that this reaction proceeded through a homolytic pathway.     

The kinetics and mechanism of oxidation of reduced spinach ferredoxin by molecular oxygen and its reduced products

Reduced spinach ferredoxin reacts with molecular oxygen in an autocatalytic reaction characterized by a hyperbolic dependence on oxygen concentration. The kinetics of the reaction indicate formation of a reduced ferredoxin-oxygen intermediate complex and production of superoxide anion which may also react with reduced ferredoxin. Hydrogen peroxide, which is formed from superoxide, in turn reoxidizes reduced ferredoxin at a rate nearly 10-times faster than that of the comparable reaction with oxygen. The kinetics of reaction of hydrogen peroxide with reduced ferredoxin are biphasic. The substrate dependence of the first phase of the reaction is consistent with a simple one-step equilibrium reaction. The second phase of the reaction could be eliminated by addition of the radical trapper, sodium formate.     

Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme

The RecBCD enzyme is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD-DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3'-end of the [5'-32P]DNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 microM to 100 microM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 microM and 25 microM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD-DNA complex that does not dissociate in the 1-2 s time-scale of our experiments.     

Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction.     

Kinetic study on the reaction mechanism of pantothenase: existence of an acyl-enzyme intermediate and role of general acid catalysis.

A kinetic study was performed on the reaction mechanism of pantothenase (EC 3.5.1.22) catalyzed hydrolysis of the pantothenic acid. A nonlinear progress curve is derived if the reaction occurs at low buffer concentrations. The nonlinearity is due to partial reversibility of the reaction; an acylenzyme (pantoyl-enzyme) is formed during the reaction, and beta-alanine, the other end product, is able to react with the acyl-enzyme and return back to pantothenate. The dependence of the beta-alanine return reaction on buffer concentration and on pH suggests a general acid catalysis during the reaction. A reaction mechanism is suggested, in which the -NH3+ form of beta-alanine participates in the return reaction, and the deacylation of the acyl-enzyme is acid catalyzed.     

Effects of Cations and Other Medium Components on the Zona-induced Acrosome Reaction of Hamster Spermatozoa

Although the acrosome reaction in lively motile hamster spermatozoa can occur independently of the egg or its investments ("spontaneous" acrosome reaction), it appears to be the egg investments, particularly the zona pellucida, that induces the acrosome reaction in fertilizing spermatozoa of many mammalian species. The latter is referred to as "zona-induced" acrosome reaction. Experiments were conducted to determine if the zona-induced acrosome reaction has different ion requirements from the spontaneous reaction. Like the spontaneous acrosome reaction, the zona-induced acrosome reaction required extracellular Na⁺, K⁺ and Ca²⁺. The absence of Cl⁻ and albumin in the medium inhibited the reaction. The zona-induced acrosome reaction could occur in a HCO₃⁻-free medium, but far less efficiently than in medium containing this ion. Proteinase inhibitors, benzamidine and TLCK, inhibited the zona-induced acrosome reaction. These results suggest that the chemical reactions involved in the spontaneous and zona-induced acrosome reactions are similar although the reaction-triggering mechanism is probably different.     

Extra-weak chemiluminescence of drugs. XII. Effect of the molar ratio of amino acid to sugar on extra-weak chemiluminescence in the Maillard reaction

The extra-weak chemiluminescence in the Maillard reaction caused by the reaction between L -lysine and D -arabinose was measured, and a linear relationship was found between the chemiluminescence and the amount of L -lysine added. After a 1-hour reaction equimolar amounts of D -arabinose and L -lysine were consumed regardless of the initial concentration of D -arabinose. The chemiluminescence of the Maillard reaction originates from Maillard reaction products formed by the equimolar reaction between sugar and amino acid and depends on the concentration of amino acid.     

Wax ester synthesis by lipase-catalyzed esterification with fungal cells immobilized on cellulose biomass support particles

Esterification reactions between long-chain alcohol and oleic acid were performed for producing wax esters. The reaction can be catalyzed efficiently by cell-bound lipase of Rhizopus niveous fungal cells immobilized within cellulose biomass support particles. Carrying out the reaction in a solvent-free system is feasible by adding a molecular sieve for dehydration purposes. To optimize the yield, addition of a molecular sieve should be performed gradually during the whole course starting from the beginning of the reaction. The influence of reaction conditions such as temperature and substrate concentrations on reaction rates and yields were investigated; however, this reaction system is under the influence of both internal and external mass transfer resistance. Conducting the reaction in an organic solvent system with hexane or heptane as the solvent can eliminate diffusional effects. Reaction kinetics were subjected to detailed study in this system. The kinetics of the reaction can be represented satisfactorily by a Ping-Pong Bi Bi mechanism with deadend inhibition by alcohol.     

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).     

Effect of mitochondria on the redox reaction between oxyhemoglobin and ferricytochrome c

The effect of mitochondria on the redox reaction between oxymyoglobin (oxy-Mb) and ferricytochrome c was studied. The parameters of this reaction in the absence of mitochondria have been investigated earlier. It is shown that the course of oxidation of oxymyoglobin by cytochrome c in the presence of mitochondria differs from that without mitochondria: no reduced cytochrome c is observed; in addition, the order of this redox reaction and its dependence on pH and ionic strength change. The factors influencing the state of mitochondrial membrane and uncouples enhance markedly the reaction rate. The conclusion was drawn that mitochondria directly participate in the oxymyoglobin-cytochrome c redox reaction. The possibility of this reaction in vivo under extreme conditions and during pathological processes is discussed.     

An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction

A moving front has been observed as a special pattern during the hydrogenase-catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thin-layer reaction chamber. Such fronts start spontaneously and at random times at different points of the reaction chamber; blue spheres are seen expanding at constant speed and amplitude. The number of observable starting points depends on the hydrogenase concentration. Fronts can be initiated by injecting either a small amount of completed reaction mixture or activated hydrogenase, but not by injecting a low concentration of reduced benzyl viologen. These characteristics are consistent with an autocatalytic reaction step in the enzyme reaction. The special characteristics of the hydrogen-uptake reaction in the bulk reaction (a long lag phase, and the enzyme concentration dependence of the lag phase) support the autocatalytic nature. We conclude that there is at least one autocatalytic reaction step in the hydrogenase-catalyzed reaction. The two possible autocatalytic schemes for hydrogenase are prion-type autocatalysis, in which two enzyme forms interact, and product-activation autocatalysis, where a reduced electron acceptor and an inactive enzyme form interact. The experimental results strongly support the occurrence of prion-type autocatalysis.     

Substrate interactions with the alpha-subunit of the Escherichia coli tryptophan synthase. A study of the activity of mutant alpha-subunits.

Extracts of 19 trpA mutant strains of Escherichia coli were examined for their relative activity in the reversible aldolytic reaction catalyzed by the trpA gene product, the α-subunit of tryptophan synthase, in combination with the β-subunit of this enzyme. The specific activities in this reaction, indoleglycerol-P (InGP) ? indole + glyceraldehyde-3-P, were determined for both the forward reaction (InGP to indole) and the reverse reaction (indole to InGP). The majority of the mutant α-subunits had <10% of the wild-type activity in the forward reaction, as expected since these mutant strains were selected for defects in this reaction. In contrast, the majority of these mutant enzymes had >50% of the wild-type activity in the reverse reaction. Several had 5 to 15% of wild-type specific activity in the forward reaction but 60 to 100% of wild-type specific activity in the reverse reaction. Spontaneous revertant strains, selected for their increased ability to catalyze the forward reaction effectively, contained α-subunits with the expected higher specific activities in the forward reaction but without parallel changes in the reverse reaction activity.     

Estimation of the contribution of the bioluminescent reaction rate and quantum yield to the enhancement of firefly bioluminescence in the presence of cationic liposomes.

Cationic liposomes containing phosphatidylcholine, cholesterol and distearyldimethylammonium chloride (DSDAC) enhanced maximum light emission (BL intensity) and total light emission from the firefly bioluminescence (BL) reaction. The increase in BL intensity was interpreted on the basis of the increase in both BL reaction rate and BL quantum yield (PhiBL) of the BL reaction. The increase in BL reaction rate was due to the increase in the localized concentration of BL reactants on the surface of cationic liposomes by electrostatic interaction. On the other hand, the increase in PhiBL was due to the change of light-emitting species in the presence of cationic liposomes. Each contribution of BL reaction rate and PhiBL to the enhancement of the BL intensity was estimated by measuring the BL reaction rate and PhiBL in the presence of cationic liposomes containing various amounts of DSDAC. The contribution of the BL reaction rate to the increase in the BL intensity was found to be two-fold greater than that of PhiBL.     

Tissue expression of proliferative antigens (PCNA and Ki-67) in oral lichen ruber related to clinical status

The aim of this study was to determine the expression intensity of PCNA and Ki-67 tissue antigens related to pathologically modified oral mucosa in OLR lesions, and to determine the reaction intensity of these antigens in individual clinical forms, i.e. lichen ruber planus (LRP) and lichen ruber erosivus (LRE) comparing the reaction intensity with the inflammation grade and the degree of hyperkeratosis in lesions of 30 patients. Control group included patients (n = 15) with oral leukoplakia simplex. Tissue antigens were observed by immunohistochemical analysis using APAAP and LSAB methods. The reaction on tested tissue antigens was focal positive and of mosaic type. The reaction of the PCNA antigen was intensely high in OLR lesions regardless on the clinical form of the lesion. The reaction intensity positively correlated with the inflammation grade and the degree of hyperkeratosis in lesions. The reaction on Ki-67 tissue antigen ranged from low to moderately high intensity. Intensely high reaction was observed in lesions of lichen ruber erosivus. The reaction positively correlated with the inflammation grade and the degree of hyperkeratosis in lesions. Observed modified reaction of analyzed tissue antigens related with individual clinical forms of OLR might be the indicator of transformed nature of these lesions.     

Triglyceride Interesterification by Lipases: 2. Reaction parameters for the reduction of trisaturated impurities and diglycerides in batch reactions

A model system consisting of pure triolein and palmitic acid and LipozymeTM, an immobilized lipase (E.C. 3.1.1.3.). has been used to determine the effects of various reaction parameters on the reaction rate and the formation of by-products in the interesterification reaction. The goal was to minimize the level of diglycerides and eliminate trisaturated triglycerides at an endpoint chosen so that the results could be applied to the production of cocoa butter substitutes. The levels of diglycerides, which are essential reaction intermediates, and trisaturated glycerides, which are believed to be formed as a result of spontaneous acyl migration of mono- and diglyceride intermediates, were determined at a defined endpoint. A lag period was observed in which no tripalmitate was formed. The content of Lipozyme used was the most powerful factor in eliminating tripalmitate formation and reducing diglycerides; by using large quantities of Lipozyme, the reaction reached the endpoint before the tripalmitate formation became measurable and low levels of diglycerides were formed. The effects of varying the ratio of palmitic acid to triolein were investigated. A complex relationship between the ratio of substrate components emerged in which the diglyceride content increased with increasing triolein concentration and the tripalmitate content was lowest at a molar ratio of palmitic acid to triolein of 3.5. The reaction was run at 70, 80, and 90°C; best results were obtained at 70°. The water activity of the reaction was adjusted prior to catalysis and maintained during the reaction by equilibrating the reaction mixture and enzyme and running the reaction in an atmosphere of controlled water activity. A direct relationship between diglycerides and water activity was observed, and the level of tripalmitate formed corresponded to the time required to reach the endpoint. The reaction system was tested using ethyl palmitate instead of palmitic acid as acyl donor; the diglyceride content again increased with increasing water activity, but larger amounts of diglycerides were formed. Much shorter reaction times were required, with small quantities of tripalmitate formed.