US 2012/0040380 A1 · OPTICALLY-DETECTABLE ENZYME SUBSTRATES AND THEIR METHOD OF USE

Abstract

The present invention relates to compounds that are substrates for an enzyme, and upon reaction with the enzyme provide a detectable response, such as an optically detectable response. In particular, the compounds have utility in detecting the presence of a β-lactamase in a sample. In addition to the compounds, methods are disclosed for analyzing a sample for the presence of a β-lactmase, for example, as an indicator of expression of a nucleic acid sequence including a sequence coding for a β-lactmase. Kits are disclosed that include the disclosed compounds and additional components, for example, cells, antibodies, a β-lactmase or instructions for using the components in an assay.

Claims26

Dependent claims

1-37. (canceled)

A compound having a structure of one of the following formulae:

where R1 is
A is S, SO, or SO2;
X is O;
L-Y is a thioether linked moiety formed by reaction with a quencher moiety having a structure of formula:
R2 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R3 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R4 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R5 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R6 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R7 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R8 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R9 is
wherein W is a dye moiety;
s is an integer from 0 to 5;
W′ is (═O);
W″ is OR10, NHR11, or halogen;
wherein R10 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl;
or substituted heteroaryl;
and,
R11 is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl;
substituted heteroaryl or OR12;
and
R12 is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl.

20 more elements

A method for determining the presence or absence of β-lactamase enzyme in a sample, the method comprising:

a) contacting the sample with a β-lactamase substrate to form a contacted sample;
b) incubating the contacted sample for a sufficient amount of time for the β-lactamase enzyme to cleave the β-lactamase substrate to form an incubated sample;
c) illuminating the incubated sample with an appropriate wavelength;
and
d) observing the illuminated sample whereby the presence or absence of β-lactamase enzyme in the sample is determined.

2 more elements

A method of localizing a fluorescent dye product in an environment comprising an aqueous solution and a β-lactamase, the method comprising:

a) contacting the environment with a non-fluorescent compound comprising a dye moiety and a β-lactam moiety;
b) incubating the product of step a) for a sufficient amount of time for the β-lactamase to cleave the dye moiety from the β-lactam moiety, thereby producing a fluorescent dye product which is insoluble in the aqueous solution, and thereby localizing the fluorescent dye product in the environment.

A method for detecting the presence or absence of a target antigen in a sample, the method comprising:

a) contacting the sample with an antibody to form a contacted sample, wherein the antibody binds either directly or indirectly to the target antigen, and wherein the antibody comprises a β-lactamase enzyme;
b) adding a non-fluorescent (β-lactamase substrate to the incubated sample to form an added sample;
c) incubating the added sample for a sufficient amount of time for the β-lactamase enzyme to cleave the non-fluorescent β-lactamase substrate to form a fluorescent dye moiety;
d) illuminating the fluorescent dye moiety with an appropriate wavelength to form an illuminated sample;
and
e) observing the illuminated sample whereby the presence or absence of the target antigen is determined.

3 more elements

A kit for determining the presence or absence of β-lactamase in a sample, the kit comprising:

a) a compound having a structure of one of the following formulae:
where R1 is
A is S, SO, or SO2;
X is O;
L-Y is a thioether linked moiety formed by reaction with a quencher moiety having a structure of formula:
R2 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R3 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R4 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R5 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R6 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R7 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R8 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R9 is
wherein W is a dye moiety;
s is an integer from 0 to 5;
W′ is (═O);
W″ is OR10, NHR11, or halogen;
wherein R10 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl;
or substituted heteroaryl;
and,
R11 is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl;
substituted heteroaryl or OR12;
and
R12 is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl;
and
b) instructions on the use of the kit.

23 more elements

Dependencies are inferred from the claim text.

Optically-detectable enzyme substrates and their method of use

Life of this patent

Optically-detectable enzyme substrates and their method of use

Life of this patent

Abstract

Claims26