OPTICALLY-DETECTABLE ENZYME SUBSTRATES AND THEIR METHOD OF USE
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.
1 . A compound having the formula:
A is S, O, SO, SO 2 or CH 2 ;
X is O, S or NH;
L is a linker;
R 2 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 3 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 4 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 5 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 6 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 7 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 8 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 9 is
in which W is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, a dye moiety or CN, and; s is an integer from 0 to 5;
W′ and W″ are independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, (═O), (═NH), OR 10 , NHR 11 , or halogen;
wherein R 10 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; or substituted heteroaryl; and,
R 11 is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; substituted heteroaryl or OR 12 ;
wherein R 12 is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl; and
Y is a dye moiety or a quencher moiety.
2 . The compound according to claim 1 , wherein the compound is other than a cephalosporin, and at least one of W, W′ or W″ is a quencher of fluorescence emitted by the first dye moiety.
3 . The compound according to claim 1 , wherein the compound is other than a cephalosporin, at least one of W, W′ and W″ includes a second dye moiety, and the first dye moiety is a quencher of the fluorescence of the second dye moiety.
4 . The compound according to claim 1 , wherein R 9 is:
where the symbol n is an integer selected from 1 to about 8.
5 . The compound according to claim 1 , wherein R 9 is other than benzyl, 2-thienylmethyl or cyanomethyl.
6 . The compound according to claim 1 , wherein the compound has the formula:
where A is S, O, SO, SO 2 or CH 2 ; one of Y and R 9 includes a dye moiety, and the other has the formula:
where W is substituted or unsubstituted alkyl substituted or unsubstituted heteroaklyl, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, or CN; the symbol s represents an integer selected from 0 to 5;
W′ and W″ are independently H, substituted or unsubstituted alkyl (═O), (═NH), OR 10 , NHR 11 , or halogen;
Wherein W, W′, and W″ are not quenchers of the fluorescence emitted by the dye moiety;
R 10 is hydrogen, alkyl substituted alkyl heteroalkyl, or substituted heteroalkyl;
R 11 is hydrogen, alkyl substituted alkyl heteroalkyl, substituted heteroalkyl, or OR 12 ;
wherein R 12 is hydrogen, alkyl substituted alkyl heteroalkyl, substituted heteroalkyl.
7 . The compound according to claim 6 , wherein R 9 is other than benzyl, 2-thienylmethyl or cyanomethyl.
8 . The compound according to claim 6 , wherein Y includes a dye moiety and R 9 is
where W is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, or CN;
s is an integer from 0 to about 5; and,
W, W′, and W″ are not quenchers of the fluorescence emitted by the dye moiety.
9 . The compound according to claim 6 , wherein R 9 is benzyl, substituted benzyl, 5-membered heteroaryl, substituted 5-membered heteroaryl, or CN.
10 . The compound according to claim 6 , wherein A is S or SO.
11 . The compound according to claim 6 , wherein, W′ is (═O) and W″ is (OH).
12 . The compound according to claim 6 , wherein R 9 is
where R 10 and R 11 are independently H, —OH, —NH 2 , substituted or unsubstituted C 1 -C 18 alkyl, substituted or unsubstituted C 1 -C 18 heteroalkyl, substituted or unsubstituted C 1 -C 18 cycloalkyl, substituted or unsubstituted C 1 -C 18 heterocycloalkyl, substituted or unsubstituted C 1 -C 18 aryl, or substituted or unsubstituted C 1 -C 18 heteroaryl.
13 . The compound according to claim 6 , wherein R 9 is other than benzyl, 2-thienylmethyl or cyanomethyl.
14 . The compound according to claim 6 , wherein the compound has the formula:
where R 9 and each R a group is independently H, substituted or unsubstituted akyl or heteroalkyl, or substituted or unsubstituted aryl or heteroaryl.
15 . The compound according to claim 1 , wherein the compound is:
16 . The compound according to claim 1 , wherein the first dye moiety is bonded to two substrate moieties, and the two substrate moieties are independently a cephalosporin, a clavulanate, an aceturate, a malonamate, benzofuranone, benzopyranone or a simple β-lactam ring substrate.
17 . The compound according to claim 16 , wherein at least one of the two substrate moieties bonded to the single dye moiety is other than a cephalosporin substrate moiety.
18 . The compound of claim 16 , wherein the compound is:
19 . The compound according to claim 16 , wherein the two substrate moieties are two clavulanates, two aceturates, two malonamates, two benzofuranones, two benzopyranones, a clavulanate and a cephalosporin, a clavulanate and an aceturate, a clavulanate and a malonamate, a clavulanate and a benzofuranone, a clavulanate and a benzopyranone, a clavulanate and a simple β-lactam ring substrate, an aceturate and a cephalosporin, an aceturate and a malonamate, an aceturate and a benzofuranone, an aceturate and a benzopyranone, an aceturate and a simple β-lactam ring substrate, a malonamate and a cephalosporin, a malonamate and a benzofuranone, a malonamate and a benzopyranone, a malonamate and a simple β-lactam ring substrate, a benzofuranone and a cephalosporin, a benzofuranone and a benzopyranone, a benzofuranone and a simple β-lactam ring substrate, a benzopyranone and a cephalosporin, or a benzopyranone and a simple β-lactam ring substrate.
20 . The compound according to claim 1 , wherein the compound is a clavulanic acid derivative (a clavulanate) and has the formula:
where Y is a first dye moiety;
L is a linker moiety;
A is S, O, SO, SO 2 , or CH 2 ;
X is O, S or NH;
W′ and W″ are independently hydrogen, alkyl substituted alkyl (═O), (═NH), OR 13 , NHR 14 ;
wherein R 13 is hydrogen, alkyl substituted alkyl heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
R 14 is hydrogen, alkyl substituted alkyl heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or OR 15 ;
wherein R 15 is hydrogen, alkyl substituted alkyl heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.
21 . The compound according to claim 20 , wherein X and A are O.
22 . The compound according to claim 21 , wherein W′ is (═O) and W″ is (OH).
23 . The compound according to claim 21 , wherein the compound has the formula:
where each R a group is independently hydrogen, alkyl substituted alkyl heteroalkyl, substituted heteroaklyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.
24 . The compound according to claim 1 , wherein the compound has the formula:
25 . 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, wherein the β-lactamase substrate has the formula:
A is S, O, SO, SO 2 or CH 2 ;
X is O, S or NH;
L is a linker;
R 2 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 3 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 4 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 5 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 6 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 7 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 8 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 9 is
in which W is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, dye moiety, or CN, and;
s is an integer from 0 to 5;
W′ and W″ are independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, (═O), (═NH), OR 10 , NHR 11 , or halogen;
wherein R 10 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; or substituted heteroaryl; and,
R 11 is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; substituted heteroaryl or OR 12 ;
wherein R 12 is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl; and
Y is a dye moiety or quencher moiety;
b) incubating the contacted sample for a sufficient amount of time for the β-lactamase enzyme to 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.
26 . 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, wherein the β-lactamase substrate has the formula:
b) incubating the contacted sample for a sufficient amount of time for the β-lactamase enzyme to 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.
27 . 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.
28 . The method according to claim 27 , wherein the non-fluorescent compound has the formula:
29 . The method according to claim 27 , wherein the environment is a member selected from a biological cell and a cell-free environment.
30 . The method according to claim 28 , wherein the biological cell environment is an interior of a cell, an interior of a cell organelle, and an exterior of a cell.
31 . 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;
e) observing the illuminated sample whereby the presence or absence of the target antigen is determined.
32 . 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, wherein the non-fluorescent β-lactamase substrate has the formula:
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;
e) observing the illuminated sample whereby the presence or absence of the target antigen is determined.
33 . A kit for determining the presence or absence of β-lactamase in a sample, the kit comprising:
a) a compound having the formula:
A is S, O, SO, SO 2 or CH 2 ;
X is O, S or NH;
L is a linker;
R 2 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 3 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 4 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 5 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 6 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 7 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 8 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
R 9 is
in which W is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, a dye moiety or CN, and; s is an integer from 0 to 5;
W′ and W″ are independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, (═O), (═NH), OR 10 , NHR 11 , or halogen;
wherein R 10 is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; or substituted heteroaryl; and,
R 11 is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; substituted heteroaryl or OR 12 ;
wherein R 12 is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl; and
Y is a dye moiety or a quencher moiety; and
b) instructions on the use of the kit.
34 . The kit according to claim 33 , further comprising at least one of a reaction buffer, a β-lactamase, a antibody conjugated to a β-lactamase enzyme, a nucleic acid sequence coding for a β-lactamase, and a cell comprising a nucleic acid sequence coding for a β-lactamase.
35 . A kit for determining the presence or absence of β-lactamase in a sample, the kit comprising:
a) a compound having the formula:
b) instructions on the use of the kit.
37 . The kit according to claim 36 , further comprising at least one of a reaction buffer, a β-lactamase, a antibody conjugated to a β-lactamase enzyme, a nucleic acid sequence coding for a β-lactamase, and a cell comprising a nucleic acid sequence coding for a β-lactamase.