PREPARATION OF S-ENANTHIOMERS OF 3-HYDROXY-1,4-BENZDIAZEPIN-2-ONE ESTERS BY BIOTECHNOLOGICAL WAY

Abstract

Aim. Improving the method of obtaining the S-enantiomer of 3-acetoxy-5-phenyl7-bromo-1,2-dihydro-3H-1,4-benzdiazepin-2-one using microsomal fraction carboxylesterase included in agar. Methods. The microsomal fraction was obtained by low-speed sedimentation in the presence of calcium ions, the protein content was determined by the Lowry-Hartree method and esterase activity - according to 1-naphthyl acetate. Obtaining a reusable biocatalyst was performed by including the microsomal fraction in agar gel. Enzymatic hydrolysis of 3-acetoxy-5-phenyl-7-bromo-1,2-dihydro-3H-1,4-benzdiazepin-2-one was performed for 2.5 h in a solution of methyl cellosolve: Na-phosphate buffer, 0.0167 M, pH 7.0 (2/3, vol./ vol.), at a temperature of 37 ºC. Enantiomeric excess was determined by HPLC. Results. The method of obtaining the S-enantiomer of 3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-benzdiazepin-2-one was improved. The inclusion of the microsomal fraction in the agar gel contributed to the development of an available method for obtaining a biocatalyst of multiple actions with high esterase activity (90%). The choice of methylcelosolve as a solvent in the enantioselective hydrolysis reaction made it possible to eliminate four stages of biotechnology for obtaining the S-enantiomer of the substrate. A modification of the carrier for column chromatography was performed. It allowed to visualize the process of separation of 1,4-benzdiazepin-2-one derivatives during chromatography using UV-irradiation. Chromatography system was changed from chloroform: 1,4-dioxane: heptane (2: 0.5: 2) to chloroform: acetonitrile (3: 0.5). This resulted in significantly better separation of 1,4-benzdiazepin-2-one derivatives. Conclusion. The method of obtaining the S-enantiomer of 3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H1,4-benzdiazepin-2-one has been improved, which made it possible to simplify the process of its isolation by 5 stages.