FLUOROGENIC ENZYME ACTIVITY ASSAY METHODS AND COMPOSITIONS USING FRAGMENTABLE LINKERS
Substrate compound-containing micelles and various compositions, kits and methods for their preparation and use are provided.
1 . A substrate compound comprising:
a) a hydrophobic moiety capable of integrating the substrate compound into a micelle;
b) a fluorescent moiety;
c) a trigger moiety; and
d) a linker linking the hydrophobic, fluorescent and trigger moieties that is capable of fragmenting to release the fluorescent moiety or the hydrophobic moiety when the trigger moiety is acted upon by a trigger agent selected from structures 4, 5, 6, and 7:
2 . The substrate compound of claim 1 in which the trigger moiety comprises a substrate for a cleaving enzyme.
3 . The substrate compound of claim 2 in which the cleaving enzyme is selected from a lipase, an esterase, a phosphatase, a protease, a glycosidase, a carboxypeptidase and a catalytic antibody.
4 . The substrate compound of claim 2 in which the linker fragments via an elimination reaction selected from the group consisting of 1,4-, 1,6-, and 1,8-elimination reactions when the substrate is cleaved from the substrate compound by the cleaving enzyme.
5 . The substrate compound of claim 4 in which the elimination is 1,4-elimination.
6 - 10 . (canceled)
11 . The substrate compound of claim 1 in which the linker fragments via elimination when the trigger moiety is acted upon by the trigger agent.
12 - 13 . (canceled)
14 . The substrate compound of claim 1 in which the fluorescent moiety comprises a fluorescent dye selected from a xanthene dye, a fluorescein dye, a rhodamine, a cyanine dye, a phthalocyanine dye, a squaraine dye and a bodipy dye.
15 . The substrate compound of claim 1 in which the hydrophobic moiety comprises a hydrocarbon group containing from 6 to 30 carbon atoms.
16 - 39 . (canceled)
40 . A micelle comprising a plurality of substrate compounds of claim 1 , each of which comprises:
a) a hydrophobic moiety capable of integrating the substrate compound into the micelle;
b) a fluorescent moiety;
c) a trigger moiety; and
d) a linker linking the hydrophobic, fluorescent and trigger moieties that is capable of fragmenting to release the fluorescent moiety or the hydrophobic moiety when the trigger moiety is acted upon by a trigger, wherein the fluorescence signals of the fluorescent moieties in the micelle are quenched as compared to their fluorescence signals when released from their respective substrate compounds.
41 . The micelle of claim 40 in which each substrate compound of the plurality is the same.
42 . The micelle of claim 40 which further comprises a plurality of quenching compounds, each of which comprises a hydrophobic moiety capable of integrating the quenching compound into the micelle and a quenching moiety capable of quenching the fluorescence signal of a fluorescent moiety in the micelle.
43 . A micelle comprising:
a) a first substrate compound of claim 1 comprising a first hydrophobic moiety capable of integrating the first substrate compound into the micelle, a first fluorescent moiety, a first trigger moiety and a first linker linking the first hydrophobic, fluorescent and trigger moieties that is capable of fragmenting to release the first fluorescent moiety or first hydrophobic moiety from the micelle when the first trigger moiety is acted upon by a first trigger; and
b) a second substrate compound of claim 1 comprising a second hydrophobic moiety capable of integrating the second substrate compound into the micelle, a second fluorescent moiety, a second trigger moiety and a second linker linking the second hydrophobic, fluorescent and trigger moieties that is capable of fragmenting to release the second fluorescent moiety or the second hydrophobic moiety from the micelle when the second trigger moiety is acted upon by a second trigger,
wherein the first and second trigger moieties are triggered by different triggers, the fluorescence signals of the first and second fluorescent moieties are resolvable from one another and the fluorescence signals of the first and second fluorescent moieties in the micelle are quenched as compared to their fluorescent signals when released from the micelle.
44 . The substrate compound of claim 3 in which the glycosidase is selected from beta-galactosidase and beta-glucoronidase.
45 . The substrate compound of claim 44 in which the glycosidase is beta-galatosidase.
46 . The substrate compound of claim 44 in which the glycosidase is beta-glucoronidase.