Method of obtaining pyroglutaminic 4-methylcoumyl-7-amide

摘要

Invention applies to bio-organic chemistry, mainly for the production of substrates of ferments used to evaluate activity of ferments and in research of their activity mechanisms. A method for obtaining fluorogenic substrate- pyroglutaminic acid 4-methylcumaril-7 amide has been developed. This substrate is used for the selective detection of pyrrolidonyl-peptidase (pyrrolidone carboxyl -peptidase), taking as a basis on fermentative truncation of 7-amino-4methylcoumarin and its ability to fluoresce at 440 nm.The method offered by the authors allows it to obtain pyroglutaminic acid 4 methylcumaril-7 amide with a high outcome (65%; in prototype-38%). The process is technologically easy (2 stages; in prototype-6): Pyroglutaminic acid is mixed in a chloroform solution with thionyl chloride, temperature: -5 to -10 C, time: 2-3 hours, chloride of pyroglutaminic acid is not eliminated from the medium; equimolar amount of 7-amino-4 methylcoumarin in absolute N, N'-dimethylformamide is added, followed by the addition of N-methylmorpholine. The mixture is kept in an ambient temperature for at least 16 hours.