CHROMOGENIC GLUCURONIDASE SUBSTRATES AND USES

摘要

Chromogenic substrates for β-D-glucuronidase activity comprising monoglucuronides of some 1,2-dihydroxyaromatic derivatives. When cleaved these form soluble coloured conjugates with multivalent metal ions such as iron ions. The substrates may be used in conjunction with chromogenic substrates for other enzymes in microbial detection and identification especially involving liquid media. Microbes can be grown in the presence of the substrates and the compounds providing the metal ion. The substrates are particularly useful for detecting β-D-glucuronidase-positive E. coli. Synthetic methods for making the compounds are described.

主权项

1. A method of detecting β-D-glucuronidase activity in a liquid medium comprising the steps of:
a) contacting a metal compound and a β-D-glucuronidase substrate according to following formula I with a substance suspected of containing or producing a glucuronidase: wherein X is independently selected from the group consisting of H, CH3, C2-C6 alkyl, a metal cation and a non-metal cation; wherein Y is independently selected from the group consisting of H, a metal cation and a non-metal cation; wherein either
i) R1 is H or OH and R4 is H or NO2; orii) R1 and R4 are independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 hydroxyalkyl, halogen, nitro, C2-C24 acyl, C2-C24 acyloxy, C7-C24 aralkyl, C6-C24 aryl, sulfonyl and amido; and wherein R2 and R3 are independently selected from the group consisting of H, OH, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 hydroxyalkyl, halogen, nitro, C2-C24 acyl, C2-C24 acyloxy, C7-C24 aralkyl, C6-C24 aryl, sulfonyl and amido provided that R2 and R3 do not both represent OH, or R2 and R3 together form the group represented by formula VI:
—CR5═CR6—CR7═CR8—  VI; wherein R5-R8 are independently selected from the group consisting of H, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 hydroxyalkyl, halogen, nitro, C2-C24 acyl, C2-C24 acyloxy, C7-C24 aralkyl, C6-C24 aryl, hydroxyl, sulfonyl and amido provided that in the formula VI any vicinal groups R5 to R8 are not both OH; such that a product of substrate cleavage is capable of chelating the metal compound, thereby forming a colored compound; and b) detecting presence of the colored compound.