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US 6,710,200 B2Granted
Cyanohydrin ethers and esters as high-sensitivity enzyme substrates
AssigneeThe Regents of the University of California
InventorsBruce D. Hammock, Guomin Shan, Rong Zhang
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Abstract
Ethers and esters of cyanohydrins that contain optically detectable moieties are highly effective for detecting, monitoring, and measuring the activity of enzymes that cause the cleavage of certain types of substrates. The cyanohydrins function as proaldehydes and proketones, spontaneously converting to aldehydes and ketones, respectively, which provide a large increase in optical detectability relative to both the starting esters and ethers and the cyanohydrins.
Claims23
A method of detecting in a sample the presence of enzyme activity that converts an ether or ester group in a substrate to an alcohol group, said method comprising:
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, or colorimety;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- and
- R3 is a group that provides said reporter with a molecular structure that upon exposure to said enzyme activity causes R3 to be replaced by H, R3 further being a member selected from the group consisting of alkyl, substituted alkyl, aralkyl, substituted aralkyl, phosphate, sulfate, carbohydrate residues, substituted carbohydrate residues, and
- in which R4 is a member selected from the group consisting of alkyl, substituted alkyl, aralkyl, substituted aralkyl, oxiranylalkyl, substituted oxiranylalkyl, oxiranylalkoxy, substituted oxiranylalkoxy, alkylamino, substituted alkylamino, peptidylamino, cycloalkylamino, substituted cycloalkylamino, oxiranylamino, substituted oxiranylamino, alkoxy, substituted alkoxy, and a residue of the acid moiety of a pyrethroid,
- under conditions by which said enzyme activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter.
8 more elements
A method for the detection of esterase activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, or colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- and
- R4 is a member selected from the group consisting of alkyl, substituted alkyl, aralkyl, substituted aralkyl, oxiranylalkyl, substituted oxiranylalkyl, oxiranylalkoxy, substituted oxiranylalkoxy, alkylamino, substituted alkylamino, peptidylamino, cycloalkylamino, substituted cycloalkylamino, oxiranylamino, substituted oxiranylamino, alkoxy, substituted alkoxy, and a residue of the acid moiety of a pyrethroid,
- R1, R2, and R4 further being selected to render said reporter a substrate for said esterase activity,
- under conditions by which said esterase activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said esterase activity.
8 more elements
A method for the detection of cytochrome P450 activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, or colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- and
- R3 is a member selected from the group consisting of alkyl, substituted alkyl, aralkyl, and substituted aralkyl,
- R1, R2, and R4 further being selected to render said reporter a substrate for said cytochrome P450 activity,
- under conditions by which said cytochrome P450 activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said esterase activity.
8 more elements
A method for the detection of epoxide hydrolase activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, or colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- R5 is a member selected from the group consisting of CH2, O, and NH;
- and
- R6 is a member selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, and substituted aralkyl,
- under conditions by which said epoxide hydrolase activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said epoxide hydrolase activity.
8 more elements
A method for the detection of glutathione S-transferase activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV or light absorptivity, or colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- under conditions by which said glutathione S-transferase activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said glutathione S-transferase activity.
5 more elements
A method for the detection of lipase activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, or colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- and
- R7 is a fatty acid residue;
- under conditions by which said lipase activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said lipase activity.
7 more elements
A method for the detection of protease activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, colorimetry;
- R2is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- and
- R7 is a polypeptide;
- under conditions by which said protease activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said protease activity.
7 more elements
A method for the detection of β-glucuronidase activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- under conditions by which said β-glucuronidase activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said β-glucuronidase activity.
5 more elements
A method for the detection of alkaline phosphatase activity in a sample, said method comprising
- (a) contacting said sample with a reporter having the formula
- in which:
- R1 is an aldehyde or ketone group that is optically detectable by fluorescence emission, UV light absorptivity, colorimetry;
- R2 is a member selected from the group consisting of H, C1-C2 alkyl, benzyl, and substituted benzyl;
- under conditions by which alkaline phosphatase activity if present would promote the conversion of said reporter to a cyanohydrin of the formula
- and the rearrangement of said cyanohydrin to a carbonyl compound of the formula
- and
- (b) detecting said carbonyl compound by optical detection selectively relative to said cyanohydrin and said reporter, and correlating said detection of said carbonyl compound with said alkaline phosphatase activity.
5 more elements
Dependencies are inferred from the claim text.