Compounds and Methods for Conjugation of Biomolecules
Low-copper click chemistry, 1.3-dipolar cycloadditions, and Staudinger ligations for modifying biomolecules is provided. Compositions, methods, and kits relating to low-copper click chemistry, 1.3-dipolar cycloadditions, and Staudinger ligations are also provided.
1 . A compound of the formula:
wherein:
A is a carbon, or A, R 5 , and R 6 are absent;
R 1 , R 2 , R 3 , and R 4 , are independently selected from hydrogen, halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally substituted one or more times by halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; or two substituents selected from R 1 , R 2 , R 3 , and R 4 , wherein each of the at least two substituents are on different carbon atoms together form a fused moiety selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, and all of the remaining substituents are independently selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl; or at least two of the remaining substituents together form a fused moiety selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, and any remaining substituents are independently selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl;
R 5 , and R 6 , are independently selected from hydrogen, halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally substituted one or more times by halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
at least one substituent selected from R 1 , R 2 , R 3 , R 4 , R 5 and R 6 comprises X-L-, wherein:
X is selected from a reporter molecule, a carrier molecule, a solid phase, a therapeutic molecule such as peptide, a protein, an antibody, a polysaccharide, a nucleic acid polymer, an ion complexing moiety, a lipid or a non-biological organic polymer or polymeric micro or nano particle, that are optionally bound to one or more additional fluorophores; or
X is a reactive group such as carboxylic acid, an activated ester of carboxylic acid, an amine, a hydrazine, a haloacetamide, an alkyl halide, an isothiocynate or a maleimide group; and
L is an independently a single covalent bond or L is covalent linkage having 1-24 non-hydrogen atoms selected from the group consisting of C, N, O, P and S and composed of any combinations of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl;
Z is an independently a single covalent bond or Z is covalent linkage having 1-10 non-hydrogen atoms selected from the group consisting of C, N, O, P and S and composed of any combinations of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and carbon-sulfur bonds in the form of a straight- or branched-chain alkyl or heteroalkyl chain; and
G is a chemical handle selected from an azide-reactive group, an alkyne-reactive group, and a phosphine-reactive group.
2 . The compound of claim 1 , wherein the compound is of the formula:
3 . The compound of claim 2 , wherein the compound is of the formula:
4 . The compound of claim 2 , wherein the compound is of the formula:
wherein
R 2 , R 3 , R 4 , and R 7 to R 12 are independently selected from hydrogen, halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally substituted one or more times by halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; or two substituents selected from R 2 , R 3 , R 4 , and R 7 to R 12 , wherein the two substituents are on different carbon atoms together form a fused moiety selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, and all of the remaining substituents are independently selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl; or two of the remaining substituents also together form a fused moiety selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, and the remaining substituents are independently selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl; or R 7 to R 12 , wherein the two substituents are on same carbon atom together form a spirocyclic moiety selected from alkyl or heteroalkyl, portions of which are further optionally substituted with halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl;
at least one substituent selected from R 1 , R 2 , R 3 , R 4 , R 5 and R 6 comprises X-L-, wherein:
X is selected from a reporter molecule, a carrier molecule, a solid phase, a therapeutic molecule such as peptide, a protein, an antibody, a polysaccharide, a nucleic acid polymer, an ion complexing moiety, a lipid or a non-biological organic polymer or polymeric micro or nano particle, that are optionally bound to one or more additional fluorophores; or
X is a reactive group such as carboxylic acid, an activated ester of carboxylic acid, an amine, a hydrazine, a haloacetamide, an alkyl halide, an isothiocynate or a maleimide group; and
L is an independently a single covalent bond or L is covalent linkage having 1-24 non-hydrogen atoms selected from the group consisting of C, N, O, P and S and composed of any combinations of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl; and
G is a chemical handle selected from an azide-reactive group, an alkyne-reactive group, and a phosphine-reactive group.
5 - 10 . (canceled)
11 . The compound of claim 1 , wherein all but one of the R substituents are H.
12 . The compound of claim 1 , wherein L is an alkyl group having a chain length of 0 to 15 atoms.
13 . The compound of claim 12 , wherein L is an alkyl group having a chain length of 0 to 5 atoms.
14 . The compound of claim 12 , wherein L is —NH—(CH 2 ) n —NH—C(O)—, wherein n is 1 to 12.
15 . The compound of claim 1 , wherein the reporter molecule comprises a chromophore, fluorophore, fluorescent protein, phosphorescent dye, tandem dye, particle, hapten, enzyme, or radioisotope.
16 . The compound of claim 15 , wherein the fluorophore is a xanthene, coumarin, cyanine, pyrene, oxazine, borapolyazaindacene, or carbopyranine.
17 . The compound of claim 15 , wherein the enzyme is horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or beta-lactamase.
18 . The compound of claim 15 , wherein the particle is a semiconductor nanocrystal.
19 . The compound of claim 1 , wherein the carrier molecule is an amino acid, peptide, protein, polysaccharide, nucleoside, nucleotide, oligonucleotide, nucleic acid, hapten, psoralen, drug, hormone, lipid, lipid assembly, tyramine, synthetic polymer, polymeric microparticle, biological cell, cellular component, ion chelating moiety, enzymatic substrate, or virus.
20 . The compound of claim 1 , wherein the carrier molecule is an antibody, antibody fragment, antigen, avidin, streptavidin, biotin, dextran, IgG binding protein, fluorescent protein, agarose, or non-biological microparticle.
21 . The compound of claim 1 , wherein the solid support is an aerogel, hydrogel, resin, bead, biochip, microfluidic chip, silicon chip, multi-well plate, membrane, conducting metal, nonconducting metal, glass, or magnetic support.
22 . The compound of claim 1 , wherein the solid support is a silica gel, polymeric membrane, particle, derivatized plastic film, glass bead, cotton, plastic bead, alumina gel, polysaccharide, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead, or starch.
23 . The compound of claim 1 , wherein the therapeutic molecule is taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, or analogs or homologs thereof.
24 . The compound of claim 1 , wherein the therapeutic molecule is an antimetabolite, alkylating agent, anthracycline, antibiotic, or anti-mitotic agent.
25 . The compound of claim 1 , wherein the therapeutic molecule is abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage colony stimulating factor, or granulocyte colony stimulating factor.
26 . The compound of claim 1 , wherein G is an alkyne-reactive group.
27 . The compound of claim 26 , wherein the alkyne-reactive group is an azide.
28 . The compound of claim 1 , wherein G is an azide-reactive group.
29 . The compound of claim 28 , wherein the azide-reactive group is a terminal alkyne.
30 - 61 . (canceled)
62 . The compound of claim 1 selected from the group consisting of:
63 . (canceled)
64 . The compound of claim 12 , which is
65 . A compound selected from the group consisting of:
66 . A composition comprising:
a compound of the formula:
wherein:
A is a carbon, or A, R 5 , and R 6 are absent;
R 1 , R 2 , R 3 , and R 4 , are independently selected from hydrogen, halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally substituted one or more times by halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; or two substituents selected from R 1 , R 2 , R 3 , and R 4 , wherein each of the at least two substituents are on different carbon atoms together form a fused moiety selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, and all of the remaining substituents are independently selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl; or at least two of the remaining substituents together form a fused moiety selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, and any remaining substituents are independently selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl;
R 5 , and R 6 , are independently selected from hydrogen, halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally substituted one or more times by halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
at least one substituent selected from R 1 , R 2 , R 3 , R 4 , R 5 and R 6 comprises X-L-, wherein:
X is selected from a reporter molecule, a carrier molecule, a solid phase, a therapeutic molecule such as peptide, a protein, an antibody, a polysaccharide, a nucleic acid polymer, an ion complexing moiety, a lipid or a non-biological organic polymer or polymeric micro or nano particle, that are optionally bound to one or more additional fluorophores; or
X is a reactive group such as carboxylic acid, an activated ester of carboxylic acid, an amine, a hydrazine, a haloacetamide, an alkyl halide, an isothiocynate or a maleimide group; and
L is an independently a single covalent bond or L is covalent linkage having 1-24 non-hydrogen atoms selected from the group consisting of C, N, O, P and S and composed of any combinations of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl;
Z is an independently a single covalent bond or Z is covalent linkage having 1-10 non-hydrogen atoms selected from the group consisting of C, N, O, P and S and composed of any combinations of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and carbon-sulfur bonds in the form of a straight- or branched-chain alkyl or heteroalkyl chain; and
G is a chemical handle selected from an azide-reactive group, an alkyne-reactive group, and a phosphine-reactive group; and
a compound of the formula:
wherein X, Y, and Z each independently have the formula:
wherein:
R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, alkylthio, alkanoylamino, alkylaminocarbonyl, arylcarboxamido, alkyl aryl portions are optionally substituted one or more times by halogen, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl, arylcarboxamido; and
B, C, D, and E are C or N; and
L 1 , L 2 , and L 3 are covalent linkage having 1-5 non-hydrogen atoms selected from the group consisting of C, N, O, P and S and composed of any combinations of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, and substituted heterocycloalkyl.