Ultra bright polymeric dyes with peptide backbones
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 , M, m 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 (I):
or a stereoisomer, salt or tautomer thereof, wherein:
M is, at each occurrence, independently a fluorescent or colored moiety comprising two or more carbon-carbon double bonds and at least one degree of conjugation;
L 1 is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups;
L 2 , L 3 , L 4 and L 5 are, at each occurrence, independently optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linkers;
R 1 is, at each occurrence, independently a natural or unnatural amino acid side chain;
R 2 and R 3 are each independently H, —OH, —SH, —NH 2 , —CO 2 H, alkyl, alkylether, alkoxy, heteroalkyl, alkylaminyl, alkylcarbonyl, alkyloxycarbonyl, Q, 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 or a linker comprising a covalent bond to a further compound of structure (I), wherein the alkyl, alkylether, alkylaminyl, alkylcarbonyl and alkyloxycarbonyl are optionally substituted with hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, or combinations thereof; Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule, a solid support or a complementary reactive group Q′;
m is, at each occurrence, independently an integer of zero or greater; and
n is an integer of one or greater.
2. The compound of claim 1 , wherein R 1 is, at each occurrence, independently H, alkyl, —CH 2 CO 2 − , —CH 2 CH 2 CO 2 − , —CH 2 CH 2 CH 2 CH 2 NH 3 + , —CH 2 CH 2 CH 2 NHC(═NH 2 + )NH 2 or imidazolyl.
3. The compound of claim 1 , wherein R 1 , L 4 and m are selected such that
has an amino acid sequence of (G) 10 , (GDGDGDGDGD) or (GKGKGKGKGK).
4. The compound of claim 1 , wherein R 1 , L 4 and m are selected such that
has an amino acid sequence capable of forming an α-helix or β-sheet secondary structure.
5. The compound of claim 4 , wherein the amino acid sequence is (GGEEFMLVYKFARKHGG) or (GGMSMVVSGG).
6. The compound of claim 1 , wherein L 4 and L 5 are absent at each occurrence.
7. The compound of claim 1 , wherein at least one occurrence of L 4 or L 5 , or both, has the following structure:
8. The compound of claim 1 , wherein at least one occurrence of L 4 or L 5 , or both, has the following structure:
9. The compound of claim 1 , wherein L′ is at each occurrence an alkylene or heteroalkylene linker.
10. The compound of claim 1 , wherein L 1 is at each occurrence a linker comprising a functional group capable of formation by reaction of two complementary reactive groups and for at least one occurrence of L 1 , the functional group can be formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thiirane functional group with a complementary reactive group.
11. The compound of claim 10 , wherein for at least one occurrence of L 1 , L 1 -M has the following structure:
wherein L 1a and L 1b are each independently optional linkers.
12. The compound of claim 10 , wherein for at least one occurrence of L 1 , L 1 -M has the following structure:
wherein L 1a and L 1b are each independently optional linkers.
13. The compound of claim 1 , wherein R 2 is —NH 2 .
14. The compound of claim 1 , wherein Q has one of the following structures:
wherein:
X is halo; and
EWG is an electron withdrawing group.
15. The compound of claim 1 , wherein one of R 2 or R 3 is a linker comprising a covalent bond to an analyte molecule, wherein the analyte molecule is a nucleic acid or a polymer thereof, amino acid or a polymer thereof, an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, prion or a linker comprising a covalent bond to a polymeric bead or non-polymeric bead.
16. The compound of claim 1 , wherein M is, at each occurrence, independently a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-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.
17. The compound of claim 1 , wherein M, at each occurrence, independently has one of the following structures:
18. The compound of claim 1 , wherein the compound has one of the following structures:
19. 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.
20. A compound having the following structure (II):
or a stereoisomer, salt or tautomer thereof, wherein:
G is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with a complementary reactive group;
L 1a , L 2 , L 3 , L 4 and L 5 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linker;
R 1 is, at each occurrence, independently a natural or unnatural amino acid side chain;
R 2 and R 3 are each independently H, —OH, —SH, —NH 2 , —CO 2 H, alkyl, alkylether, alkoxy, heteroalkyl, alkylaminyl, alkylcarbonyl, alkyloxycarbonyl, Q, 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 or a linker comprising a covalent bond to a further compound of structure (I), wherein the alkyl, alkylether, alkylaminyl, alkylcarbonyl and alkyloxycarbonyl are optionally substituted with hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, or combinations thereof;
Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule, a solid support or a complementary reactive group Q′;
m is, at each occurrence, independently an integer of zero or greater; and
n is an integer of one or greater.