Toshiyuki Furutani

n-acylation of β-amino alcohol by acyl migration following enzyme-catalyzed esterification

Abstract Lipase-catalyzed n -acylations of β-amino alcohols such as ethanolamine and l -serine were investigated. To prepare n -acyl derivatives by taking advantage of the acyl migration, we first carried out a screening of suitable enzymes for the desired reaction. As a result, we found a higher activity for n -acylation with Lipase Q L. This lipase had higher hydrolytic activity for the o-acyl compound but not the n -acyl compound. The observation shows that n -acylation results from the esterification and successive acyl migration into the amino group. Using Lipase Q L, we then investigated the n -acylation of ethanolamine or l -serine with fatty acids as acyl donors. The reaction parameters for the n -acylation were clarified.

Simple screening method for lipase for transesterification in organic solvent

We investigated lipase-catalyzed hydrolysis in water and dioxane—water with a simple colorimetric method. We screened 24 lipases for the ability to hydrolyze p-nitrophenyl esters as chromogenic substrates. Their hydrolytic activities were varied by adding dioxane. Most of the lipases showed high activity in hydrolysis in water, but some showed activity in 50% dioxane—water several tens times higher than those in water. Moreover, several lipases with hydrolytic abilities in 50% dioxane—water also catalyzed the transesterification of p-nitrophenol using fatty acid vinyl esters. We found it possible that a useful lipase for transesterification can be selected by measuring the hydrolysis activity of p-nitrophenyl ester in 50% dioxane—water.

Preparation of n-, o-diacyl ethanolamine from n-acyl ethanolamine using lipase preparations

Abstract Lipase-catalyzedo-acylations of n -acyl ethanolamines were investigated in organic solvent. Of 23 lipase (esterase) preparations, half of the enzymes catalyzed the myristoylation of n -myristoyl ethanolamine in hexane. Reaction conditions for acylation were investigated using lipases such as Lipase QL from Alcaligenes sp., Lipase PS from Pseudomonas sp., and Lipase AK-20 from Pseudomonas sp. which were selected as lipases with higher activities for o -acylation. The most suitable organic solvents studied for o -acylation were hexane and toluene in the case of myristic acid as the acyl donor and acetone and tetrahydrofuran in the case of vinyl myristate as an acyl donor. The appropriate concentration of water was 0.5–1%; addition of more water-reduced the acylation activity of the enzyme because of the reverse reaction. The reaction rate for acylation was accelerated with the fatty acid vinyl ester as an acyl donor. The effective chain length of the acyl donor for o -acylation of n -myristoyl ethanolamine was around C 14 C 18 alkyl chains in free acids but was C 2 C 14 in vinyl esters. The optimum acyl chain length of n -acyl ethanolamine for myristoylation of n -acyl ethanolamine was altered by the acyl donor and solvent. In the case of racemic 3-hydroxymyristic acid as the free acyl donor for acylation of n -myristoyl ethanolamine using Lipase QL, the resulting compound was only n -myristoyl- o -3-hydroxymyristoyl ethanolamine and the e.e. value of the resulting compound was 50% of the ( R ) conformation. Lipase QL proved to be useful for acylation using optically active fatty acid.

A membrane bioreactor combined with crystallizer for production of optically active (2R, 3S)-3-(4-methoxyphenyl)-glycidic acid methyl ester

Abstract The combination of a membrane bioreactor using lipase from Serratia marcescens and a crystallizer has been proposed for the production of optically active ( 2R, 3S )-3-(4-methoxyphenyl) glycidic acid methyl ester ((−)MPGM) from racemic compounds ((±)MPGM). The reaction kinetics were investigated with a view toward industrial application of this newly devised bioreactor system. The following results were obtained. (i) The hydrolysis of (±)MPGM in the bioreactor system proceeded as a first-order reaction with respect to the substrate amount. (ii) The reaction rate depended on the amount of enzyme loaded onto a membrane module and the initial amount of substrate in the crystallizer. (iii) The reaction rate was not influenced by the rate of substrate circulation between the crystallizer and membrane bioreactor. (iv) Although the level of performance of the bioreactor system with respect to hydrolysis decreased with increasing operation time, it recovered to upon loading of fresh enzyme onto the membrane. This bioreactor system in which an enzymatic reaction and product crystallization occurred simultaneously had a high level of productivity compared with that of the conventional membrane bioreactor using a solubilized (±)MPGM substrate.

Inhibition of Octapeptide N-Myristoylation by Acyl Amino Acids and Acyl Alkanolamines

Several acyl amino acids and acyl alkanolamines were prepared and screened for their inhibition of octapeptide N-myristoylation and HIV-1 replication in MT-4 cells. Of the 62 acyl derivatives tested, N-myristoyl-O-caproyl-L-serine, N-myristoyl-O-caproyl-D-serine and N-decanoyl-O-myristoyl-L-serine were found to be uncompetitive inhibitors of N-myristoylation, but did not prevent HIV-induced cytopathicity in MT-4 cells. However, other acyl derivatives such as N-3-hydroxymyristoyl ethanolamine, N-3-hydroxymyristoyl-D-serine and N-myristoyl-L-cysteine, which did not inhibit N-myristoylation, suppressed the cytopathicity in the infected cells. The acyl derivatives described here may serve as lead compounds for antiviral agents.

Production of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester using an emulsion bioreactor containing lipase fromSerratia marcescens

An emulsion bioreactor for production of (2R,3S)-3-(4-methoxyphenyl) glycidic acid methyl ester ([-]MPGM) from a racemic mixture ([±]MPGM) using the lipase fromSerratia marcescens has been proposed. Kinetics of hydrolyzing reaction and purification of (-)MPGM from the reaction mixture were investigated to provide a basis for industrial application of this bioreactor. The hydrolyzing reaction in the bioreactor proceeded at a rate that was first order in substrate concentration. The reaction rate was affected by a stirring speed and the ratio of the aqueous phase containing lipase to the toluene phase containing substrate. Phase separation after the enzymatic reaction was accomplished by addition of surfactant to the reaction mixture, and crystalline (-)MPGM with a chemical purity of 100% and optical purity of 100% enantiomeric excess was obtained in a high yield of 40–43% by concentration of the toluene solution.