Covalent modification of biological macromolecules

• US 9,988,433 B2
• Filed: 03/13/2014
• Issued: 06/05/2018
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Mosaic Biosciences, Inc.
• Original Assignee: Mosaic Biosciences, Inc.
• Inventors: Kazantsev, Alexei, Mariner, Peter D., Stanton, Martin
• Primary Examiner(s): ROBINSON, HOPE A
• Application Number:
  US14/210,106
• Publication Number:
  US 20140273153A1
• Time in Patent Office: 1,545 Days
• Field of Search: None
• CPC Class Codes: A61K 47/557 the modifying agent being biotin
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/75 Fibrinogen
• CPC Class Codes: C12N 9/63 derived from plants
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 304/22006 Chymopapain (3.4.22.6)

Abstract

The present disclosure provides a method of covalently modifying a biological macromolecule, the method comprising subjecting a reaction mixture comprising: (a) a biological macromolecule comprising one or more thiol groups; and (b) a molecule comprising one or more olefin or alkyne moieties to a radical reaction under conditions sufficient to produce the covalently modified biological macromolecule. The present disclosure also provides a method of covalently modifying a biological macromolecule, the method comprising subjecting a reaction mixture comprising: (a) a molecule comprising one or more thiol groups; and (b) a biological macromolecule comprising one or more olefin or alkyne moieties to a radical reaction under conditions sufficient to produce the covalently modified biological macromolecule. The present disclosure further provides a covalently modified biological macromolecule prepared by any of the disclosed methods. The covalently modified biological macromolecules may be further crosslinked to form a scaffold.

Claims

1. A method of covalently modifying a fibrinogen protein, comprising:

(I) subjecting a fibrinogen protein to denaturing and reducing conditions to produce a reduced, denatured fibrinogen protein comprising one or more thiol groups;

(II) reacting the reduced, denatured fibrinogen protein with one or more norbornene moieties under

conditions sufficient to produce a photoinitiated radical thiol-ene coupling reaction between at least one of the one or more norbornene moieties and at least one of the one or more thiol groups on the reduced, denatured fibrinogen protein, thereby providing a covalently modified fibrinogen protein,

wherein the one or more norbornene moieties comprise a polymer selected from the group consisting of;

poly(lactic acid) (PLA), polyglycolide (PGA), copolymer of PLA and PGA (PLGA), poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), poly(ethylene oxide), poly(ethylene oxide)-co-poly(propylene oxide) block copolymer, poloxamine, polyanhydride, polyorthoester, poly(hydroxy acid), polydioxanone, polycarbonate, polyaminocarbonate, poly(vinyl pyrrolidone), poly(ethyl oxazoline), carboxymethyl cellulose, and hydroxyalkylated cellulose.

2. The method of claim 1, wherein the photoinitiated radical thiol-ene coupling reaction is performed under denaturing conditions.

3. The method of claim 1, wherein the polymer is poly(ethylene glycol) (PEG).

4. The method of claim 1, wherein the one or more norbornene moieties is a linear PEG-dinorbornene.

5. The method of claim 4, wherein the linear PEG-dinorbornene has an average molecular weight of 3.4 kDa.

6. The method of claim 4, wherein the reduced, denatured fibrinogen protein is reacted with a stoichiometric excess of linear PEG-dinorbornene.

7. The method of claim 1, wherein the denaturing conditions comprise dissolving the fibrinogen protein in a buffered urea solution.

8. The method of claim 1, wherein the reducing conditions comprise treatment with the reducing agent tris(2-carboxyethyl)phosphine.

9. The method of claim 8, wherein the method further comprises removing unreacted tris(2-carboxyethyl)phosphine from the reduced, denatured fibrinogen prior to step (II).

10. The method of claim 1, wherein the conditions sufficient to produce a photoinitiated radical thiol-ene coupling reaction comprise a photo-initiator.

11. The method of claim 10, wherein the photo-initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

12. The method of claim 1, wherein the photoinitiated radical thiol-ene coupling reaction is essentially completed within 5 minutes of radical initiation.

13. The method of claim 1, wherein the photoinitiated radical thiol-ene coupling reaction is essentially completed within 1 minute of radical initiation.

14. The method of claim 11, wherein the conditions sufficient to produce a photoinitiated radical thiol-ene coupling reaction comprise irradiating with light at a wavelength of 385 nm.

15. The method of claim 1, further comprising refolding the covalently modified fibrinogen protein.

16. The method of claim 1, further comprising reacting the covalently modified fibrinogen protein with a multifunctional thiol containing crosslinker.

17. The method of claim 16, wherein the multifunctional thiol containing crosslinker is dithiothreitol.