Protein labeling with cyanobenzothiazole conjugates
View Patent ↗The invention provides compounds and methods for site-specifically labeling proteins with cyanobenzothiazole derivatives of formula I. For example, the invention provides methods for labeling the N-terminus of a protein that terminates with a cysteine residue. The invention also provides methods for isolating an N-terminally labeled protein and methods for detecting an N-terminally labeled protein.
1. A method for labeling a peptide or protein, comprising:
contacting a sample comprising an N-terminal cysteine labeled peptide or protein with a cyanobenzothiazole derivative of Formula I
wherein
Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylic acid salt, alkyl amide, phosphate, alkyl phosphonate, sulfate, alkyl sulfonate, nitro, or C 1 -C 10 alkyl optionally unsaturated and optionally substituted with amino, hydroxy, oxo (═O), nitro, thiol, or halo;
each R 1 is independently H, F, Cl, Br, I, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkyl sulfonate, or CO 2 M wherein M is H, an organic cation, or an inorganic cation;
n is 0, 1, or 2;
Y is absent or is a linking group comprising C 1 -C 16 alkyl optionally substituted with one or more halo, oxo (═O), C 1 -C 6 alkyl, or C 1 -C 6 alkoxy, and optionally interrupted with one or more N(R 1 ), O, S, or —NH—C(═O)— groups; and
X is a fluorescent dye;
so as to yield a fluorescently-labeled peptide or protein.
2. The method of claim 1 , further comprising detecting the fluorescently-labeled peptide or protein.
3. The method of claim 1 , wherein the sample comprises a cell-free translation system.
4. The method of claim 1 , wherein the sample comprises intact eukaryotic cells.
5. The method of claim 1 , wherein the peptide is synthetically derived.
6. The method of claim 1 , further comprising:
combining the fluorescently-labeled peptide or protein with a sample comprising a second peptide or protein that interacts, or is suspected of interacting with, the fluorescently-labeled peptide or protein, wherein the interaction yields a complex; and
detecting fluorescence in the complex,
wherein the second protein is a fusion protein.
7. The method of claim 1 , wherein the N-terminal cysteine residue of the peptide or protein is produced by protease cleavage.
8. The method of claim 7 , wherein the protease is TEV protease.
9. The method of claim 6 , wherein the detection is performed using FRET or BRET.
10. A method for detecting a peptide or protein, comprising
a) contacting a sample comprising an N-terminal cysteine labeled peptide or protein with a cyanobenzothiazole derivative of Formula I
wherein
Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylic acid salt, alkyl amide, phosphate, alkyl phosphonate, sulfate, alkyl sulfonate, nitro, or C 1 -C 10 alkyl optionally unsaturated and optionally substituted with amino, hydroxy, oxo (═O), nitro, thiol, or halo;
each R 1 is independently H, F, Cl, Br, I, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkyl sulfonate, or CO 2 M wherein M is H, an organic cation, or an inorganic cation;
n is 0, 1, or 2;
Y is a linking group comprising C 1 -C 16 alkyl optionally substituted with one or more halo, oxo (═O), C 1 -C 6 alkyl, or C 1 -C 6 alkoxy, and optionally interrupted with one or more N(R 1 ), O, S, or —NH—C(═O)— groups; and
X is a fluorescent dye;
so as to yield a fluorescently-labeled peptide or protein; and
b) detecting the presence or absence of fluorescence in the sample.
11. The method of claim 10 , wherein the sample comprises a cell-free translation system.
12. The method of claim 10 , wherein the sample comprises intact eukaryotic cells.
13. The method of claim 10 , wherein the peptide is synthetically derived.
14. The method of claim 10 , wherein the N-terminal cysteine residue of the peptide or protein is produced by protease cleavage.
15. The method of claim 14 , wherein the protease is TEV protease.
16. The method of claim 10 , further comprising:
combining the fluorescently-labeled peptide or protein with a sample comprising a second peptide or protein that interacts, or is suspected of interacting with, the fluorescently-labeled peptide or protein, wherein the interaction yields a complex; and
detecting fluorescence in the complex.
17. A method for detecting a peptide or protein, comprising:
a) contacting a sample comprising an N-terminal cysteine labeled peptide or protein with a cyanobenzothiazole derivative of Formula I
wherein
Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylic acid salt, alkyl amide, phosphate, alkyl phosphonate, sulfate, alkyl sulfonate, nitro, or C 1 -C 10 alkyl optionally unsaturated and optionally substituted with amino, hydroxy, oxo (═O), nitro, thiol, or halo;
each R 1 is independently H, F, Cl, Br, I, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkyl sulfonate, or CO 2 M wherein M is H, an organic cation, or an inorganic cation;
n is 0, 1, or 2;
Y is a linking group comprising C 1 -C 16 alkyl optionally substituted with one or more halo, oxo (═O), C 1 -C 6 alkyl, or C 1 -C 6 alkoxy, and optionally interrupted with one or more N(R 1 ), O, S, or —NH—C(═O)— groups; and
X is a fluorescent dye;
so as to yield a fluorescently-labeled peptide or protein; and
b) combining the fluorescently-labeled peptide or protein with a sample comprising a second peptide or protein that interacts, or is suspected of interacting with, the fluorescently-labeled peptide or protein, wherein the interaction yields a complex; and
c) detecting fluorescence in the complex.
18. Amend as follows:
The method of claim 17 , wherein the sample of step
a) comprises a cell-free translation system.
19. Amend as follows:
The method of claim 17 , wherein the sample of step
a) comprises intact eukaryotic cells.
20. The method of claim 17 , wherein the peptide is synthetically derived.
21. The method of claim 17 , wherein the N-terminal cysteine residue of the peptide or protein is produced by protease cleavage.
22. The method of claim 21 , wherein the protease is TEV protease.
23. The method of claim 17 , wherein the detection is performed using FRET or BRET.