Protein labeling with cyanobenzothiazole conjugates

Patent metadata

Field	Value
Publication number	US-9932365-B2
Application number	US-201715623663-A
Country	US
Kind code	B2
Filing date	Jun 15, 2017
Priority date	Mar 27, 2008
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Promega Corp

Inventors

• Kelts Jessica
• Meisenheimer Poncho
• Shultz John

Classifications

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title

• C07D493/10

  Spiro-condensed systems · CPC title

• C07D495/04

  Ortho-condensed systems · CPC title

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C09B23/083

  five >CH- groups · CPC title