Protein labeling with cyanobenzothiazole conjugates

Patent metadata

Field	Value
Publication number	US-9689868-B2
Application number	US-201615281460-A
Country	US
Kind code	B2
Filing date	Sep 30, 2016
Priority date	Mar 27, 2008
Publication date	Jun 27, 2017
Grant date	Jun 27, 2017

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 immobilizing a protein of interest, comprising:
a) contacting a sample comprising an N-terminal cysteine labeled protein of interest 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 alkylene optionally substituted with one or more halo, oxo (═O), C r O 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 solid support;
and
b) immobilizing the protein of interest.

2. The method of claim 1 , wherein the protein of interest is purified.

3. The method of claim 1 , wherein the solid support is a particle, resin, bead, or surface.

4. The method of claim 1 , wherein the solid support comprises a silicate, polymer, sepharose, cellulose, alginate, polystyrene, a membrane, or glass.

5. The method of claim 1 , wherein the solid support is paramagnetic or magnetic.

6. The method of claim 1 , wherein the protein of interest maintains its functionality upon immobilization.

7. The method of claim 1 , wherein immobilization provides site-specific immobilization of the protein of interest.

8. The method of claim 7 , wherein the site-specific immobilization occurs at the N-terminal cysteine of the protein of interest.

Assignees

• Promega Corp

Inventors

• Kelts Jessica
• Meisenheimer Poncho
• Shultz John

Classifications

• C09B23/083

  five >CH- groups · CPC title

• C07K1/13 Primary

  Labelling of peptides · 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

• C07D495/04

  Ortho-condensed systems · CPC title

• G01N33/533 Primary

  with fluorescent label · CPC title