Stepwise synthesis of oligopeptides with N-carboxy alpha-amino acid anhydrides

Oligopeptides were synthesized in high yields by the controlled coupling reaction of N-carboxy α-amino acid anhydrides (NCA's) with amino acid and peptide sodium salts in the heterogeneous reaction medium of acetonitrile–water which contained sodium carbonate. In this solvent, it could be considered that the reaction could occur at the interface of acetonitrile and aqueous layers, and that NCA's could be protected by the organic layer from side reactions such as hydrolysis and polymerization. The careful control of reaction conditions such as pH of the solution was not necessary when the heterogeneous mixed solvent was used. Sodium carbonate which had not been used for a reaction of this kind could be satisfactorily used for stabilizing the carbamate intermediates in the aqueous layer of the heterogeneous reaction medium.     

Kinetic resolution of alpha-methylbenzylamine with omicron-transaminase screened from soil microorganisms: application of a biphasic system to overcome product inhibition

Two microorganisms showing high omicron-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-alpha-methylbenzylamine (alpha-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of omicron-transaminases were investigated. omicron-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of alpha-MBA and showed remarkable stability. However, omicron-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of alpha-MBA. The reaction rate of omicron-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (Vorg:Vaq = 1:4) in the biphasic system with cyclohexanone, the reaction rate of omicron-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM alpha-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-alpha-MBA with ca. 51% conversion. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 348-358, 1997.     

Thermolysin and alpha-chymotrypsin mediated synthesis of tripeptides containing proline

The tripeptides Z-Pro-Leu-Gly-OEt, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OBzl were synthesized by thermolysin and alpha-chymotrypsin catalysis. The optimum conditions for the couplings between Z-Pro-OH and H-Leu-OEt or H-Leu-Gly-OEt catalysed by thermolysin were determined by a systematic study involving analysis of pH effect, ammonium sulfate concentration, reaction time, enzyme concentration, and relative proportion of the carboxyl and amine components. The best yield obtained for Z-Pro-Leu-OEt was 77% and for Z-Pro-Leu-Gly-OEt, 100%. Z-Pro-Leu-OEt was coupled to H-Gly-OEt, H-Gly-NH2 and H-Gly-OBzl. The best conditions to obtain Z-Pro-Leu-Gly-OEt and Z-Pro-Leu-Gly-NH2 were determined by the study of some factors that affect the reaction yield, such as organic solvent presence, substrate ratio and aqueous and organic solvent ratio. The yield obtained under optimum synthesis conditions was 55% for Z-Pro-Leu-Gly-OEt and 61% for Z-Pro-Leu-Gly-NH2. Z-Pro-Leu-Gly-OBzl was synthesized with 42% yield.     

Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Synthesis of heteroaromatic N-beta-glycosides of N-acetylghicosamine under phase transfer conditions. III. 1,2,4-triazoIin-3-ones glucosaminides

The catalytic phase transfer reactions of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl chloride with thiosubstituted 4-phenyl-5-R-thio-Delta2-1,2,4-triazolin-3-ones in solid alkali-organic solvent and in aqueous alkali-organic solvent were studied. The main products ofglucosaminilation reaction are the appropriate N-beta-glucosaminides. The effect of reaction conditions on the yield and composition of products formed was elucidated on the example of the reaction alpha-D-glucosaminyl chloride with 5-methylthio-4-phenyl-Delta2-1,2,4-triazolin-3-one. The optimal conditions of phase transfer glycosylation were established. N-1,2-trans-grycosidic bond formation was proved by 1H NMR and IR data as well as a comparison with spectral data obtained previously for the glycosides of similar structure.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.     

Water-stable all-biodegradable microparticles in nanofibers by electrospinning of aqueous dispersions for biotechnical plant protection

Pheromone eluting oligolactide (OLA) microcapsules immobilized in electrospun biodegradable polyester nanofibers were obtained by electrospinning of aqueous dispersions of the microcapsules. OLA was prepared by conventional melt polycondensation of lactic acid. Following the protocol of the solvent displacement method, OLA was dissolved in acetone and mixed with Brij S20 and the pheromone of the European grape vine moth, Lobesia Botrana, (E,Z)-7,9-dodecadien-l-yl acetate (DA). Up to 32 wt % of this mixture could be dispersed in water with colloidal stability of several weeks without any sedimentation. Without DA as well as OLA, no stable dispersions of OLA in water were obtained. Replacement of DA by classical hydrophobes typically used for miniemulsions did not yield stable dispersions, but the addition of octyl acetate, which shows structural similarity to DA, yielded stable dispersions in water up to 10 wt %. Dispersions of OLA/DA were successfully electrospun in combination with an aqueous dispersion of a biodegradable block copolyester resulting in water-stable nanofibers containing OLA/DA microcapsules. Release of DA from microcapsules and fibers was retarded in comparison with non-encapsulated DA, as shown by model studies.     

Microbial synthesis of ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate by asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate in an aqueous-organic solvent biphasic system

In this study, whole cells of Saccharomyces uvarum SW-58 were applied in an aqueous-organic solvent biphasic system for the asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate to ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate [(R)-2]. The results of reduction in different aqueous-organic solvent biphasic systems showed that dibutylphthalate provided the best compromise between the biocompatibility and the partition of substrate and product among the solvents tested. To optimize the reaction, several factors such as reaction pH, temperature, shaking speed, volume ratio of the aqueous phase to the organic phase and ratio of biomass/substrate were investigated. It was found that the change of these factors obviously influenced the conversion and initial reaction rate, and had a minor effect on the enatiomeric excess of the product. Under the optimal conditions, 85.0% of conversion and 85.2% of enatiomeric excess were achieved. The bioconversion in the biphasic system was more efficient compared with that in the monophasic aqueous system, and product concentration as high as 54.6 g/L was reached in the organic phase without addition of co-enzyme.     

Microbial oxidation of naphthalene to cis-1,2-naphthalene dihydrodiol using naphthalene dioxygenase in biphasic media

The selective oxidation of aryl substrates to chiral cis-1,2-dihydrodiols is an industrially important reaction for the production of intermediates that can be used to produce fine chemicals, pharmaceuticals, and many other bioactive natural products. More specifically, the oxidation of naphthalene to produce optically pure (+)-cis-(1R,2S)-1,2-napthalene dihydrodiol (NDHD) to be used as a chiral synthon for specialty chemicals has gained much interest. Escherichia coli JM109(DE3) pDTG141 expresses naphthalene dioxygenase which catalyzes this reaction. Poor substrate solubility and substrate toxicity are barriers to using the power of these enzymes in large-scale aqueous whole cell systems. A biphasic reaction system was chosen to overcome these barriers. The optimal biphasic conditions for E. coli JM109(DE3) pDTG141 were determined to be 20% dodecane as the organic solvent containing 40 g/L naphthalene. The productivity of the biotransformation using resting cells was 1.75 g-diol/g-cdw/h for the first 6 h with 20% organic phase, which was increased from 0.59 g-diol/g-cdw/h for growing cells with 40% organic phase. The biocatalytic activity was retained for at least 12 h. The biocatalyst could be recycled for at least four runs in both suspended and immobilized form. The stability of the 12 h recycle was improved by immobilization in calcium alginate beads. The process has been improved both environmentally and economically by reducing the amount of solvent used and by recycling the biocatalyst.     

Kinetics of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester

We studied kinetics of thermolysin-catalyzed peptide synthesis in an aqueous/organic biphasic system theoretically and experimentally. As a model reaction producing a condensation product having no dissociating groups, we used the synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe2OMe) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-phenylalanine methyl ester (PheOMe). Usually, ethyl acetate was used as the organic solvent. First we studied the kinetics of the synthesis of Z-Phe2OMe in a buffer solution saturated with ethyl acetate. Then, factors that may affect the kinetics in the biphasic system were examined. The course of Z-Phe2OMe synthesis in the biphasic system was explained by the rate equations obtained, using the partitions of substrate and product and non-enzymatic decomposition of PheOMe. In the biphasic reaction system, the rate of synthesis was lower for a wide range of pH due to the unfavorable partition of PheOMe in the aqueous phase, but yields were higher than in the buffer solution. The effects of the organic solvents on the rate of synthesis could also be explained by variations in the partition coefficient of PheOMe. Finally, we gave a way to predict the aqueous-phase pH change caused by partitioning of the substrate. The significance of the pH change was shown in connection with the reaction using the immobilized enzyme in an organic solvent.     

Kinetically controlled synthesis of ampicillin and cephalexin in highly condensed systems in the absence of a liquid aqueous phase

Advantages of performing penicillin G amidase catalysed synthesis of ampicillin and cephalexin by enzymatic acyl transfer to the β-lactam antibiotic nuclei in a highly condensed system using mainly undissolved substrates, with no apparent aqueous liquid phase, were demonstrated. It was shown that synthesis can be performed in the absence of a liquid phase formed by water or an organic co-solvent. This highly condensed system is formed by a liquid phase given by one of the reactant, the phenylglycine methyl ester (PGM), that remains liquid in these operative conditions and the partially dissolved β-lactam nucleus. Operating in such highly condensed system, the water that causes the hydrolysis of PGM is limited to the water hydrating the support on which the enzyme is covalently immobilised. In this way the reaction system is maintained at a controlled degree of hydration.

In the present work the reaction system was modulated by eliminating the solvent (aqueous or aqueous/organic), reducing the amount of water to the minimum for the biocatalytic activity and using PGM as solvent and reagent at the same time. The synthesis was conducted with equimolar amounts of PGM and the β-lactam nucleus, with a reduced hydrolysis of the activated acyl donor. We have also studied a simple and efficient method for the workup of the reaction where the unreacted reagents can be recovered after selective filtration and precipitation.     

低共熔溶剂对酶的影响及应用研究*

低共熔溶剂是由一定化学计量比的氢键受体和氢键供体组合而成的新型绿色溶剂,具有成本低、易制备、环境友好等特点,可以作为普通有机溶剂和离子液体的替代溶剂。酶作为生物催化剂时反应条件温和,对反应底物专一性高,并且具有极高的催化效率和反应速度。酶促反应通常发生在水溶液体系,但近年来发现在低共熔溶剂中酶促反应也能有效进行。综述酶与低共熔溶剂共同作用的机理以及低共熔溶剂在酶促反应中的应用,展望未来的研究方向,为酶促反应体系的进一步开发奠定理论基础。     

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).     

Factor affecting enzyme characteristics of bilirubin oxidase suspensions in organic solvents

The activity of bilirubin oxidase toward bilirubin was studied in a liquid/solid two-phase low-water organic system using a simple spectrophotometric assay to follow the reaction. The enzyme was lyophilized from aqueous solution before being suspended in the organic solvent reaction medium. The activity was significantly influenced by the properties of the aqueous medium from which the enzyme was lyophilized, specifically its pH, and the quantity and nature of the buffering species. Analyses of these effect showed that the role of buffering species in such systems went beyond their effect in fixing the protonation state of the enzyme. The activity was also influenced by the quantity of water added to the organic solvent reaction medium. The reaction was shown to follow Michaelis-Menten Kinetics, and K(m) and k(cat) were determined. The liquid/solid two-phase system studied was extensively compared to a previously studied water-in-oil microemulsion system (c) 1993 Wiley & Sons, Inc.     

Influence of reaction conditions on the oxidation of thiol groups in model peptidomimetic oligoureas

Working with thiols is associated with the risk of unwanted oxidation reactions, which includes the formation of disulfides. Here, the use of short peptidomimetic oligoureas as a model system to study the influence of reaction conditions on the thiol oxidation process is described. The formation of disulfide bond is usually performed in aqueous solutions; in this study, it was shown that the oxidation reaction occurs also in organic medium. It was discovered that the rate of the oxidation reaction strongly depends on the type of solvent, which is the consequence of the change of the oxidant (air or DMSO), the temperature, and the presence (or absence) of base. The details reported here may help those working with thiols to find suitable conditions to control the oxidation behaviour in organic solvents, ensuring that ─SH groups either remain intact or form disulfide bonds as desired.     

Temperature optima of enzyme-catalysed reactions in microemulsion systems

Ternary phase systems (water/surfactant/organic solvent) were utilised to increase and broaden the temperature optima of enzyme-catalysed reactions. Alcohol dehydrogenases from yeast and Thermoanaerobium brockii (EC 1.1.1.1 and EC 1.1.1.2), lactate dehydrogenase from Lactobacillus delbrueckii (EC 1.1.1.28) and the particulate hydrogenase from Ralstonia eutropha (EC 1.18.99.1) were used as model enzymes in microemulsions, consisting of the surfactant Aerosol OT, and various alkane solvent and aqueous phases. All enzymes exhibited, besides an increase in specific activity, an upshift of the temperature optimum of the catalysed reaction. The temperature optimum could be further shifted by variation of the chain length of the solvent used and/or the addition of compatible solutes to the aqueous phase. Under optimised conditions, catalytic reactions of enzymes from mesophilic microorganisms had temperature optima in the range generally obtained with enzymes from thermophilic organisms.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 &#117 h.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 λh.     

Papain-catalyzed polymerization of amino acids in low water organic solvents

Summary Polyethylene glycol-modified papain catalyzed the oligomerization of amino acid amides in toluene. The water content of the organic solvent was a critical factor determining the extent of polymerization. Under optimized conditions, lysine and phenylalanine oligomers containing up to 10 residues were obtained. In sharp contrast to what is observed in aqueous media, hydrophilic and basic amino acid derivatives resulted in higher reaction yields than hydrophobic amino acid derivatives.