Hydrolase enzyme substrates and uses thereof

摘要

The present invention provides novel methods for determining the presence or amount of a hydrolytic enzyme in a sample, based on novel substrates for the enzymes, and also provides compositions and methods that provide highly sensitive assay methods for such hydrolytic enzymes.

主权项

1. A method for assessing activity and/or amount of a hydrolytic enzyme in a sample, which method comprises:
a) contacting a sample containing or suspected of containing a hydrolytic enzyme with a hydrolytic enzyme substrate having a structure of formula (I): under conditions where said hydrolytic enzyme, if present in said sample, cleaves said substrate to produce an aryl alcohol molecule or unsaturated aliphatic alcohol molecule having a structure of formula (II) and a compound having a structure of formula (III): wherein A is an aromatic or heteroaromatic group, a 1-alkene or a 1-alkyne, each of which is optionally substituted; each R is independently H or an optionally substituted C1-C4 alkyl or aryl; n is an integer from 1-4; and X is a group comprising a substrate moiety; and b) assessing the presence and/or amount of said aryl alcohol molecule or unsaturated aliphatic alcohol molecule to assess activity and/or amount of said hydrolytic enzyme in said sample, wherein the step of assessing the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule comprises oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with enzymatic oxidation in the presence of oxygen to produce H2O2 and assessing the presence and/or amount of the H2O2, or enzymatic oxidation in the presence of NAD+ or NADP+ to produce NADH or NADPH, and assessing the presence and/or amount of the NAD+, NADP+, NADH or NADPH.