Ionic polymers comprising fluorescent or colored reporter groups
Polymers comprising first and second fluorescent or colored moieties on the polymer backbone and two or more charged moieties on the polymer backbone between the first and second colored or fluorescent moieties are provided. The polymers are useful as fluorescent or colored dyes. Methods associated with preparation and use of such polymers are also provided.
1. A polymer comprising:
i) a backbone;
ii) two or more positively charged moieties on the backbone; and
iii) first and second colored or fluorescent moieties on the backbone,
wherein the two or more positively charged moieties are in a position on the polymer backbone between the first and second colored or fluorescent moieties, and having the following structure (I):
or a stereoisomer, salt or tautomer thereof, wherein:
M is, at each occurrence, independently the fluorescent or colored moiety;
P 1 is, at each occurrence, independently one of the following structures:
wherein:
R is H or C 1 -C 6 alkyl;
x is an integer from 0 to 6; and
m is an integer of 1 or greater, provided that m is selected such that P 1 comprises at least two positively charged moieties;
P 2 is, at each occurrence, independently a section of the backbone to which the fluorescent or colored moiety is attached;
L 1 is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups, wherein for at least one occurrence of L 1 , L 1 -M has one of the following structures:
wherein L 1a and L 1b are each independently optional linkers;
L 2 is at each occurrence, independently absent, a section of the backbone or a linker linking P 2 to R 2 or P 2 to R 3 ;
L 3 is, at each occurrence, independently an optional linker linking P 1 to P 2 ;
R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(R b )R c , Q or L′;
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 sulfhydryl, disulfide, N-succinimide ester, imidoester, polyflourophenyl ester, isothiocyanate, alkyl azide, an acyl azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, maleimide functional group, or a moiety selected from one of the following structures:
wherein:
X is halo; and
EWG is an electron withdrawing group;
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); and
n is an integer of one or greater.
2. The polymer of claim 1 , wherein L 2 and L 3 are, at each occurrence, independently C 1 -C 6 alkylene, C 2 -C 6 alkenylene or C 2 -C 6 alkynylene.
3. The polymer of claim 1 , wherein R 2 and R 3 are each independently OH or —OP(═R a )(R b )R c .
4. The polymer of claim 1 , wherein one of R 2 and R 3 is OH or —OP(═R a )(R b )R c , and the other of R 2 and R 3 is Q or a linker comprising a covalent bond to Q.
5. The polymer of claim 1 , wherein one of R 2 and R 3 is OH or —OP(═R a )(R b )R c , and the other of R 2 and R 3 is a linker comprising a covalent bond to a nucleic acid or polymer thereof, an amino acid or polymer thereof, an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer or prion or a linker comprising a covalent bond to a polymeric bead or non-polymeric bead.
6. The polymer of claim 1 , wherein the fluorescent or colored moiety of M is, at each occurrence, independently a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, bis-fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9, 10-ethynylanthracene or ter-naphthyl moiety.
7. The polymer of claim 1 , wherein the fluorescent or colored moiety of M is, at each occurrence, independently p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide or a derivative of perylene, acridine, cyanine, perylene imide, or perylene amide.
8. The polymer of claim 1 , wherein the fluorescent or colored moiety of M is, at each occurrence, independently a coumarin dye, resorufin dye, dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energy transfer dye, thiazole orange dye, polymethine or N-aryl-1,8-naphthalimide dye.
9. The polymer of claim 1 , wherein the fluorescent or colored moiety of M is, at each occurrence, independently pyrene, perylene, perylene monoimide or 6-FAM or a derivative of pyrene, perylene, or perylene monoimide.
10. The polymer of claim 1 , wherein the fluorescent or colored moiety of M is, at each occurrence, independently has one of the following structures:
11. A polymer having one of the following structures:
wherein:
x is, at each occurrence, independently an integer of 1 or greater, provided that x is selected such that the compound includes at least two positively charged moieties for at least one integral value of y;
y is an integer of 2 or greater;
R 2 , R 3 and L 1 are defined according to claim 1 ; and
F is a fluorescein moiety having the following structure:
12. A method of staining a sample, comprising adding to said sample the polymer of claim 1 in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.
13. The method of claim 12 , wherein said optical response is a fluorescent response, the sample comprises cells, and the method further comprises observing the cells by flow cytometry.
14. A method for visually detecting an analyte molecule, the method comprising:
(a) providing the polymer of claim 1 , wherein the polymer comprises a covalent bond to the analyte molecule; and
(b) detecting the polymer by its visible properties.