Compounds and methods for conjugation of biomolecules

Patent metadata

Field	Value
Publication number	US-10648985-B2
Application number	US-201816107762-A
Country	US
Kind code	B2
Filing date	Aug 21, 2018
Priority date	Mar 4, 2011
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

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.

First claim

The invention claimed is:

1. A compound of the formula:

wherein:
A is a carbon;
R 1 , R 2 , R 4 , R 5 , and R 6 , are hydrogen;
R 3 comprises X-L-, wherein:
X is selected from a reporter molecule, a carrier molecule, a solid support, and a reactive group, that are optionally bound to one or more additional fluorophores, wherein:
the reporter molecule comprises a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme, or a radioisotope;
the carrier molecule is an amino acid, a peptide, a protein, an antibody, an antibody fragment, an antigen, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a biological cell, a cellular compartment, an ion chelating moiety, an enzymatic substrate, or a virus;
the solid support is an aerogel, a hydrogel, a resin, a silica gel, a bead, a biochip, a microfluidic chip, a silicon ship, a multi-well plate, a membrane, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, alumina gel, polysaccharide, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, a conducting metal, a nonconducting metal, glass, magnetic bead, paramagnetic bead, superparamagnetic bead, or a magnetic support;
the reactive group is a 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 NH—(CH 2 ) n —NH—C(O)—, wherein n is 1 to 12;

Z is a single covalent bond; and
G is an azide.

2. The compound of claim 1 , wherein the fluorophore is a xanthene, coumarin, cyanine, pyrene, oxazine, borapolyazaindacene, or carbopyranine.

3. The compound of claim 1 , wherein the enzyme is horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or beta-lactamase.

4. A method of modifying a biomolecule comprising the step of reacting in a solution a biomolecule comprising an azide reactive moiety with a compound of claim 1 to provide a modified biomolecule.

5. The method of claim 4 , wherein the azide reactive moiety comprises a terminal alkyne, an activated alkyne, or triarylphosphine.

6. The method of claim 4 , wherein the biomolecule is a nucleic acid, oligonucleotide, protein, peptide, carbohydrate, polysaccharide, glycoprotein, lipid, hormone, drug, or prodrug.

7. The method of claim 4 , wherein the solution further comprises copper ions.

8. The method of claim 7 , wherein the method further comprises at least one reducing agent.

9. The method of claim 7 , wherein the method further comprises a copper chelator.

10. A kit comprising a compound of claim 1 .

11. The kit of claim 10 , wherein the kit further comprises a copper ion source.

12. The kit of claim 10 , wherein the kit further comprises at least one reducing agent.

13. The kit of claim 10 , wherein the kit further comprises a copper chelator.

14. A compound selected from the group consisting of:

Assignees

• Life Technologies Corp

Inventors

• Gee Kyle
• Singh Upinder
• Grecian Scott
• Clarke Scott

Classifications

• C07D213/79

  Acids; Esters · CPC title

• C07D231/12

  with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms · CPC title

• C07D213/75 Primary

  Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates · CPC title

• C07D213/74

  Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals · CPC title

• C07D213/81

  Amides; Imides · CPC title