Polymeric tandem dyes with linker groups
Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I): or a stereoisomer, tautomer or salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , M 1 , M 2 , m and n are as defined herein. Methods associated with preparation and use of such compounds is also provided.
1. A compound having the following structure (I):
or a stereoisomer, salt or tautomer thereof, wherein:
M 1 and M 2 are, at each occurrence, independently a fluorescent or colored chromophore, provided that at least one of M 1 and M 2 is a FRET donor, and another one of M 1 and M 2 is a corresponding FRET acceptor, at least one of the FRET donor chromophores is positioned between two FRET acceptor chromophores, and at least one occurrence of M 1 and M 2 are different chromophores selected such that M 1 and M 2 form a FRET pair;
L 1 is, at each occurrence, independently an optional alkylene or heteroalkylene linker;
L 2 and L 3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;
L 4 is, at each occurrence, independently an alkylene or heteroalkylene linker;
R 1 is, at each occurrence, independently H, alkyl or alkoxy;
R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(Rdb)R c , Q, or a protected form thereof, or L′;
R 4 is, at each occurrence, independently OH, SH, O − , S − , OR d or SR d ;
R 5 is, at each occurrence, independently oxo, thioxo or absent;
R a is O or S;
R b is OH, SH, O − , S − , OR d or SR d ;
R c is OH, SH, O − , S − , OR d , OL′, SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;
R d is a counter ion;
Q is, at each occurrence, independently a moiety comprising a reactive group, or protected form thereof, capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support or a complementary reactive group Q;
L′ is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (I);
m is, at each occurrence, independently an integer of zero or greater; and
n is an integer of two or greater,
provided that at least one occurrence of L 4 is heteroalkylene or at least one occurrence of m is 0 when M 1 and M 2 are selected from fluorescein, pyrene and perylene chromophores.
2. The compound of claim 1 , wherein the compound has the following structure (IA):
wherein:
z is, at each occurrence, independently an integer from 1 to 100; and
m 1 , m 2 and m 3 are, at each occurrence, independently an integer from 0 to 6.
3. The compound of claim 1 , wherein the compound has the following structure (IB):
wherein:
z is, at each occurrence, independently an integer from 1 to 100.
4. The compound of claim 1 , wherein the compound has the following structure (IC):
wherein:
x 1 , x 2 , x 3 and x 4 are, at each occurrence, independently an integer from 0 to 6.
5. The compound of claim 2 , wherein the compound has the following structure (ID):
6. The compound of claim 1 , wherein the compound has the following structure (IE):
7. A compound having the following structure (II):
or a stereoisomer, salt or tautomer thereof, wherein:
M 1 and M 2 are, at each occurrence, independently a fluorescent or colored chromophore, provided that at least one occurrence of M 1 and M 2 are different chromophores, at least one occurrence of M 1 and M 2 combine to form a FRET acceptor-donor pair, and the FRET donor chromophore in the FRET acceptor-donor pair is positioned between two FRET acceptor chromophores;
L 1 is at each occurrence, independently an optional alkylene or heteroalkylene linker;
L 2 and L 3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;
L 4 is, at each occurrence, independently an alkylene or heteroalkylene linker;
R 1 is, at each occurrence, independently H, alkyl or alkoxy;
R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′;
R 4 is, at each occurrence, independently OH, SH, O − , S − , OR d or SR d ;
R 5 is, at each occurrence, independently oxo, thioxo or absent;
R a is O or S;
R b is OH, SH, O − , S − , OR d or SR d ;
R c is OH, SH, O − , S − , OR d , OL′, SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;
R d is a counter ion;
Q is, at each occurrence, independently a moiety comprising a reactive group, or protected form thereof, capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support or a complementary reactive group Q′;
L′ is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (II);
m is, at each occurrence, independently an integer of zero to two or greater than 6; and
n is an integer of two or greater.
8. The compound of claim 1 , wherein L 1 has one of the following structures:
wherein
a, b, and c are each independently an integer ranging from 1-6.
9. The compound of claim 1 , wherein R 4 is, at each occurrence, independently OH, O − or OR d , R 5 is, at each occurrence, oxo, and R 1 is, at each occurrence, H.
10. The compound of claim 1 , wherein R 2 and R 3 are each independently —OP(═R a )(R b )R c .
11. The compound of claim 10 , wherein R c is OL′.
12. The compound of claim 11 , wherein L′ is a heteroalkylene linker to: Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I).
13. The compound of claim 12 , wherein L′ comprises an alkylene oxide or phosphodiester moiety, or combinations thereof.
14. The compound of claim 11 , wherein L′ has the following structure:
wherein:
m″ and n″ are independently an integer from 1 to 10;
R e is H, an electron pair or a counter ion;
L″ is R e or a direct bond or linkage to: Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I).
15. The compound of claim 1 , wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional group.
16. The compound of claim 1 , wherein Q has one of the following structures:
wherein each X is independently a halogen.
17. The compound of claim 1 , wherein R 2 or R 3 has one of the following structures:
18. The compound of claim 1 , wherein M 1 and M 2 at each occurrence, independently have one of the following structures:
19. The compound of claim 1 , wherein the compound has one of the following structures:
wherein HEG, R 3 , dT, B, B′, C, F, T and Y have the following structures, respectively:
20. The compound of claim 7 , wherein the compound has one of the following structures:
wherein F and Y have the following structures, respectively:
21. A method of staining a sample, comprising adding to said sample the compound of claim 1 in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.
22. A method for visually detecting an analyte molecule, the method comprising:
(a) providing the polymer compound of claim 1 , wherein the polymer compound comprises a covalent bond to the analyte molecule; and
(b) detecting the polymer compound by its visible properties.
23. A method for visually detecting an analyte molecule, the method comprising:
(a) ad-mixing the polymer compound of claim 1 , wherein the polymer compound comprises a covalent bond to Q, with the analyte molecule;
(b) forming a bio-conjugate of the polymer compound and the analyte molecule; and
(c) detecting the bio-conjugate by its visible properties.