Protein derivatization to endow cell penetration

Patent metadata

Field	Value
Publication number	US-10258695-B2
Application number	US-201514845108-A
Country	US
Kind code	B2
Filing date	Sep 3, 2015
Priority date	Sep 4, 2014
Publication date	Apr 16, 2019
Grant date	Apr 16, 2019

Abstract

Methods and reagents for enhancing cellular uptake of a cargo molecule by covalently bonding optionally-substituted fluorenyl groups to the cargo molecules, where cellular uptake includes at least partial uptake into the cytosol. Useful fluorenylation reagents include those of formula:

and salts thereof where variables are as defined. Cargo molecules include peptides and proteins. Also provided are fluorenylated cargo molecules, including fluorenylated peptides and proteins.

First claim

The invention claimed is:

1. A method for increasing cellular uptake of a protein by fluorenylating the protein by covalently bonding one or more optionally substituted fluorenyl groups to the protein and contacting a cell with the fluorenylated protein, wherein cellular uptake of the fluorenylated protein is increased compared to the protein without fluorenylation, wherein fluorenylation is carried out by esterification of one or more carboxylate groups of the protein.

2. The method of claim 1 wherein fluorenylation is carried out by esterification of one or more carboxylate groups of the protein with a diazofluorene of formula I:

or salts thereof, wherein:
R 3 -R 10 are selected from hydrogen, alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy, aryl, aryl oxy, alkylaryl, alkylaryloxy, arylalkyl, arylalkyloxy, heteroaryl, heteroaryloxy, carbocyclic, carbocyclyloxy, heterocyclic or heterocyclyloxy groups each of which is optionally substituted; or
R 3 -R 10 are selected from non-hydrogen substituents selected from halogens, hydroxyl, nitro groups, cyano, isocyano, thiocyano, isothiocyano, sulfuryl, —N(R′) 2 , —COR′, —COOR′, —CON(R′) 2 , —NR′—CO—R′, —NR′—CO—N(R′) 2 —, —CO—SR′, —SO 2 —NR′ 2 , —OR′, or —SR′, where each R′, independently, is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic groups, each of which groups is optionally substituted, or two of R 3 -R 10 are linked together to form an optionally substituted carbocyclic, aryl, heterocyclic or heteroaryl ring wherein one or two carbons of the ring are optionally replaced with —CO— and the carbocyclic or heterocyclic rings are saturated or unsaturated.

3. The method of claim 2 , wherein the compound of formula I is 9-diazofluorene.

4. The method of claim 1 , wherein the protein is an antibody or functional fragment thereof or is an enzyme.

5. The method of claim 4 , wherein fluorenylation is carried out by esterification of one or more carboxylate groups of the protein with 9-diazofluorene.

6. The method of claim 1 , which is carried out in vitro.

7. The method of claim 1 , wherein the protein retains at least 10% of its biological activity on fluorenylation compared to the protein without fluorenylation.

8. The method of claim 1 , wherein two or more carboxylate groups of the protein are fluorenylated.

9. The method of claim 1 , wherein the fluorenylated protein is taken up at least in part into the cytosol of the cell.

10. The method of claim 9 , wherein after uptake into the cell, the fluorenyl groups of the fluorenylated protein are removed within the cell.

11. The method of claim 1 , wherein fluorenylation is carried out by esterification of one or more carboxylate groups of the protein with a diazofluorene.

12. The method of claim 1 , wherein increasing cellular uptake represents an enhancement of 2-fold or higher of cellular uptake of the fluorenylated protein compared to cellular uptake of the corresponding protein that is not fluorenylated.

13. The method of claim 9 , wherein increasing cellular uptake represents an enhancement of 2-fold or higher of cellular uptake into the cytosol of the fluroenylated protein compared to cellular uptake into the cytosol of the protein that is not fluorenylated.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Raines Ronald T
• Andersen Kristen A

Classifications

• C07K1/13

  Labelling of peptides · CPC title

• C07K1/1077

  by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids · CPC title

• A61K47/54 Primary

  the modifying agent being an organic compound · CPC title