Water soluble fluorescent or colored dyes and methods for their use
Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I): including stereoisomers, salts and tautomers thereof, wherein R 1 , R 2 , R 3 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , M 1 , M 2 , A, q, w and n are as defined herein. Methods associated with preparation and use of such compounds are also provided.
1. A compound having the following structure (III):
wherein:
M 1 and M 2 are, at each occurrence, independently a moiety comprising two or more double bonds and at least one degree of conjugation, and at least one occurrence of M 1 is a moiety comprising three or more aryl or heteroaryl rings, or combinations thereof;
L 1 , L 3 , L 4 L 6 , L 7 and L 8 are, at each occurrence, independently optional alkylene or heteroalkylene linkers;
R 1 is, at each occurrence, independently H, alkyl or alkoxy;
R 2 is an electron pair, H, alkyl, alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, phospho, thiophospho, alkylphospho, alkylthiophospho, alkyletherphospho, alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkyl or thiophosphoalkylether, or R 2 is a linker comprising a covalent bond to a biomolecule or microparticle;
R 3 is H, OH, SH, —NH 2 , alkyl, alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate, alkylphospho, alkylthiophospho, —Oalkylphospho, —Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho, —Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl, phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether, —Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether;
R 4 is, at each occurrence, independently O − , S − , OZ, SZ or N(R 6 ) 2 , where Z is a cation and each R 6 is independently H or alkyl;
R 5 is, at each occurrence, independently oxo, thioxo or absent;
n is an integer from 2 to 20; and
q and w are each independently 0 or 1 for each integral value of n, wherein q is 1 for at least two integral values of n, or wherein q and w are each independently one for at least one integral value of n.
2. The compound of claim 1 , wherein the compound has the following structure (IIa):
wherein:
M 1 is, at each occurrence, independently a moiety comprising two or more double bonds and at least one degree of conjugation, and at least one occurrence of M 1 is a moiety comprising four or more aryl or heteroaryl rings, or combinations thereof;
L 1 , L 7 and L 3 are, at each occurrence, independently optional alkylene or heteroalkylene linkers;
R 1 is, at each occurrence, independently H, alkyl or alkoxy;
R 2 is H, alkyl, alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, phospho, thiophospho, alkylphospho, alkylthiophospho, alkyletherphospho, alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkyl or thiophosphoalkylether, or R 2 is a linker comprising a covalent bond to a biomolecule or microparticle;
R 3 is H, OH, SH, —NH 2 , alkyl, alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate, alkylphospho, alkylthiophospho, —Oalkylphospho, —Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho, —Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl, phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether, —Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether;
R 4 is, at each occurrence, independently O − , S − , OZ, SZ or N(R 6 ) 2 , where Z is a cation and each R 6 is independently H or alkyl;
R 5 is, at each occurrence, independently oxo, thioxo or absent; and
n is an integer from 2 to 10.
3. The compound of claim 1 , wherein the compound has one of the following structures (IIc), (IId), (IIe) or (IIf):
4. The compound of claim 1 , wherein the compound has the following structure (IIIb):
wherein:
R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are, at each occurrence, independently H or alkyl; and
x 1 , x 2 , y 1 , y 2 , z 1 and z 2 are, at each occurrence, independently an integer from 0 to 5.
5. The compound of claim 1 , wherein R 2 has one of the following structures:
wherein:
R 2a is —OH, —NH 2 , or —SH; and
a is an integer from 1 to 10.
6. The compound of claim 1 , wherein R 2 has one of the following structures:
wherein:
R 2a is —OH, —SH, —NH 2 , phosphate or thiophosphate;
R 4a and R 4b are independently O − , S − , OZ or SZ, where Z is a cation;
R 5a and R 5b are independently oxo, or thioxo; and
a, b and c are each independently integers from 1 to 10.
7. The compound of claim 1 , wherein R 3 is OH.
8. The compound of claim 1 , wherein R 3 has one of the following structures:
wherein:
R 3a is —OH, —SH, —NH 2 , phosphate or thiophosphate;
R 4a and R 4b are independently O − , S − , OZ or SZ, where Z is a cation;
R 5a and R 5b are independently oxo, or thioxo; and
b and c are each independently integers from 1 to 10.
9. The compound of claim 1 , wherein at least one M 1 is a moiety comprising four or more aryl or heteroaryl rings, or combinations thereof.
10. The compound of claim 1 , wherein M 1 is, at each occurrence, independently fluorescent or colored.
11. The compound of claim 1 , wherein M 1 has one of the following structures:
12. The compound of claim 1 , wherein R 2 is a linker comprising a covalent bond to a biomolecule or microparticle, and R 3 is H, OH, phosphate, thiophosphate, phosphoalkyl, —Ophosphoalkyl, phosphoalkylether, —Ophosphoalkylether, thiophosphoalkyl, —Othiophospho, thiophosphoalkylether or —Othiophosphoalkylether.
13. A compound having one of the following structures:
14. A method of staining a sample, comprising adding to said sample the compound of claim 12 in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.
15. A method for visually detecting a biomolecule, the method comprising:
(a) providing the compound of claim 12 ; and
(b) detecting the compound by its visible properties.
16. A method for visually detecting a biomolecule, the method comprising:
(a) admixing the compound of claim 1 with one or more biomolecules; and
(b) detecting the compound by its visible properties.
17. A composition comprising the compound of claim 1 and one or more biomolecules.
18. The compound of claim 1 , wherein R 2 is hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl or sulfhydrylalkylether.
19. The compound of claim 1 , wherein R 2 is alkylphospho, alkylthiophospho, alkyletherphospho, alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkyl or thiophosphoalkylether, wherein R 2 is optionally substituted with a substituent selected from —OH, —NH 2 , and —SH.
20. The compound of claim 1 , wherein R 3 is, phosphate, thiophosphate, phospho, thiophospho, —Oalkylphospho, —Oalkylthiophospho, —Oalkyletherphospho, —Oalkyletherthiophospho, —Ophosphoalkyl, —Ophosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether optionally substituted with a substituent selected from —OH, —NH 2 , and —SH.
21. The compound of claim 1 , wherein n is an integer from 2 to 15.
22. The compound of claim 1 , wherein each M 1 is pyrene, perylene, perylene monoimide or 6-FAM or derivative thereof.
23. The compound of claim 1 , wherein R 2 is a linker comprising a covalent bond to a biomolecule.
24. The compound of claim 23 , wherein the biomolecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer or prion.
25. The method of claim 14 , wherein said optical response is a fluorescent response.
26. The method of claim 14 , wherein said sample comprises cells.
27. The method of claim 26 , further comprising observing said cells by flow cytometry.