Pro-fluorescent probes
View Patent ↗The present invention provides a novel class of pro-fluorescent probes for reactive oxygen species (ROS). One exemplary probe is mitochondria peroxy yellow 1 (MitoPY1), a new type of flurophore for imaging mitochondrial H 2 O 2 in living cells with ROS and spatial specificity. The invention also provides methods of using pro-fluorescent probes to detect analytes. One exemplary method comprises using a pro-fluorescent probe of the invention to detect an explosive.
1. A pro-fluorescent compound having the formula:
wherein
X and Z are members independently selected from CR 5 R 6 , C(O), NR 5 , O and substituted or unsubstituted heterocycloalkyl
wherein R 5 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 6 is a member selected from CN, COR′, OR 8 , substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl
wherein R 7 is a member selected from OR 9 and NR 9 R 10
wherein R 9 and R 10 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 8 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
r is an integer selected from 1, 2 and 3;
R 1 , R 2 , R 3 and R 4 are members independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
L 1 is a linker which is a member selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and
X 1 is a member selected from a charged group, NR 11 R 12 , OR 13 , substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl
wherein R 11 , R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
2. The compound according to claim 1 , having the formula:
3. A compound having the formula:
wherein
A and E are members independently selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
X is a member independently selected from CR 5 R 6 , C(O), NR 5 , O and substituted or unsubstituted heterocycloalkyl
wherein R 5 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 6 is a member selected from CN, COR′, OW, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl wherein R 7 is a member selected from OR 9 and NR 9 R 10
wherein R 9 and R 10 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 8 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 1 , R 2 , R 3 and R 4 are members independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
L 1 is a linker which is a member selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and
X 1 is a member selected from a charged group, NR 11 R 12 , OR 13 substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl
wherein R 11 , R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
X 2 is a member selected from NR 14 , O and S;
R 14 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a linker covalently attached to a reactive functional group and a linker covalently attached to a carrier; and
G is a member selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
4. The compound according to claim 3 wherein G is substituted by —C(O)OH.
5. The compound according to any preceding claim wherein L 1 is a bond and X 1 is a member selected from NR 11 R 12 and OR 13 .
6. The compound according to claim 5 wherein at least one of R 11 and R 12 is H.
7. The compound according to claim 6 wherein both R 11 and R 12 is H.
8. The compound according to claim 5 wherein R 13 is H.
9. The compound according to claim 1 , wherein the charged group is a member selected from —OPO 3 2− , —P + R′R″R′″, —N + R′R″R′″, —S + R′R″ and —C(O)O − , wherein R′, R″ and R′″ are each independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
10. A compound which is a member selected from:
wherein R is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
11. A compound, having the formula:
wherein A and E are members independently selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, wherein at least one of A and E is substituted with a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
X and Z are members independently selected from CR 5 R 6 , C(O), NR 5 , O and substituted or unsubstituted heterocycloalkyl
wherein R 5 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 6 is a member selected from CN, COR 7 , OR 8 , substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl
wherein R 7 is a member selected from OR 9 and NR 9 R 10
wherein R 9 and R 10 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
R 8 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;
n is an integer selected from 0 and 1;
r is an integer selected from 1, 2 and 3;
R 1 , R 2 , R 3 and R 4 are members independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
L 1 is a linker which is a member selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and
X 1 is a member selected from a charged group, NR 11 R 12 , OR 13 , substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl
wherein R 11 , R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
12. The compound according to claim 11 , wherein said member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl with which a member selected from A and E is substituted bears a reactive functional group at the ω-position.
13. The compound according to claim 11 , wherein said substituted or unsubstituted heteroalkyl comprises a polyether.
14. The compound according to claim 13 , wherein said polyether is a member selected from ethylene glycol, ethylene glycol oligomers and combinations thereof, wherein said polyether has a molecular weight of from about 60 daltons to about 10,000 daltons.
15. The compound according to claim 11 , wherein said heteroalkyl is a member selected from ω-carboxyl alkyl groups, ω-carboxyl substituted alkyl groups and combinations thereof.
16. The compound according to claim 11 , wherein said substituted alkyl or substituted heteroalkyl is an active ester of a member selected from said ω-carboxyl alkyl groups, ω-carboxyl substituted alkyl groups and combinations thereof.
17. The compound according to claim 15 , wherein said ω-carboxyl substituted alkyl group has the formula:
wherein X O is a member selected from O, S and NR 15
wherein R 15 is a member selected from H, alkyl and substituted alkyl;
Y is a member selected from OR 16 and a halogen;
R 16 is a member selected from a single negative charge, H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and
j and k are members independently selected from the group consisting of integers from 1 to 20.
18. The compound according to claim 1 , wherein said compound is covalently attached to a carrier species.
19. The compound according to claim 18 , wherein said carrier species is a member selected from the group consisting of synthetic polymers and biomolecules.
20. The compound according to claim 19 , wherein said biomolecule is a member selected from the group consisting of antibodies, antigens, peptides, nucleic acids, enzymes, haptens, carbohydrates and pharmacologically active agents.
21. A microarray comprising a compound according to claim 1 , said compound being conjugated directly to a solid support, conjugated through a linker, or conjugated to a carrier species attached to said solid support.
22. The microarray according to claim 21 , wherein said carrier species is a member selected from a biomolecule, a synthetic polymer and combinations thereof.
23. The microarray according to claim 21 , wherein said solid support is divided into a first region and a second region, said first region having attached thereto a first said compound and said second region having attached thereto a second said compound.
24. A method for assaying a sample for a peroxide, said method comprising:
(a) contacting said sample with a pro-fluorescent compound according to claim 1 , 10 or 11 , under conditions appropriate to convert said compound to a fluorescent compound;
(b) exciting said fluorescent compound; and
(c) determining a fluorescence property of said sample, wherein the presence of said analyte in said sample results in a change in said fluorescence property.
25. The method according to claim 24 wherein said analyte converts said pro-fluorescent compound into said fluorescent compound.
26. The method according to claim 24 wherein said peroxide is an organic peroxide or inorganic peroxide.
27. The method according to claim 24 wherein said peroxide does not have the structure R—O—O—R′ wherein R and R′ are independently selected from H and CH 3 .