# US 11,439,711 B2 · Degradable thiol-ene polymers and methods of making thereof

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- Number: US 11,439,711 B2
- Document ID: US11439711B2
- Status: Granted
- Kind: B2
- Application number: 17/085,798
- Filed: 2020-10-30
- Published: 2021-05-27
- Granted: 2022-09-13
- Earliest claimed: 2015-02-09
- Assignees: Mosaic Biosciences, Inc.
- Applicants: Mosaic Biosciences, Inc.
- Inventors: Alexei V. Kazantsev; Peter D. Mariner; Martin Stanton
- Web page: https://patentgrep.com/patent/US11439711B2

## CPC

- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 9/06: Ointments; Bases therefor; {Other semi-solid forms, e.g. creams, sticks, gels (composition of ointments, creams or gels A61K47/00)}
- A61K 38/08: Peptides having 5 to 11 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C08B 37/0069: {Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof}
- C08B 37/0075: {Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof}
- C08G 65/3342: {having sulfur bound to carbon and hydrogen}
- C08G 75/045: from mercapto compounds and unsaturated compounds
- C08G 75/12: Polythioether-ethers (C08G75/0245 takes precedence)
- C08G 81/00: Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers (involving only carbon-to-carbon unsaturated bond reactions C08F299/00 {; polyester-amides C08G69/44; polyester-imides C08G73/16; polyamides-imides C08G73/14; block- or graft polymers containing polysiloxane sequences C08G77/42})
- C08H 1/00: Macromolecular products derived from proteins (food proteins A23; glue, gelatine C09H)
- C08J 3/075: Macromolecular gels
- C08J 3/246: {Intercrosslinking of at least two polymers}
- C08L 101/16: the macromolecular compounds being biodegradable
- C12N 9/2462: {Lysozyme (3.2.1.17)}
- C12Y 302/01017: Lysozyme (3.2.1.17)
- C08G 2210/00: Compositions for preparing hydrogels
- C08G 2650/04: End-capping
- C08G 2650/16: Photopolymerisation
- C08G 2650/20: Cross-linking
- C08J 2300/16: Biodegradable polymers
- C08J 2371/02: Polyalkylene oxides
- C08J 2389/00: Characterised by the use of proteins; Derivatives thereof
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08H: DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS (polysaccharides C08B; natural rubber C08C; natural resins or their derivatives C09F; bituminous materials C10)
- C08J: WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H (working, e.g. shaping, of plastics B29)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Provided are methods for linking polypeptides (including peptides and proteins) to other moieties using radical imitated thiol-ene chemistries, for example, modifying a polypeptide by introducing reactive thiol groups and reacting the thiol groups with olefin-containing reagents or alkyne-containing reagents under conditions that support radical thiol-ene or thiol-yne reactions. The reactive thiol groups have greater activity for radical thiol-ene reactions that a cysteine thiol group, including thiol groups that are separated from the peptide backbone by at least two carbon atoms, for example, the thiol group of a homocysteine residue. Also provided are compositions and biomaterials containing the linked polypeptides, for example, peptide and protein conjugates, and thiol-ene based biocompatible hydrogel polymers, and their uses in the medical field.

## Claims

### Claim 1

A method for selectively linking a polypeptide in the presence of one or more cysteine residues,
wherein the polypeptide comprises a peptide backbone comprising a homocysteine residue having a reactive thiol group or a 2-amino-5-mercaptopentanoic acid residue having a reactive thiol group,
wherein the method comprises reacting the reactive thiol group of the homocysteine residue or the 2-amino-5-mercaptopentanoic acid residue of the polypeptide with an ene compound comprising one or more reactive ene groups under conditions that promote a radical-mediated thiol-ene reaction.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the radical-mediated thiol-ene reaction is initiated with a radical initiator.

### Claim 3 (depends on 1)

The method according to claim 1, wherein comprises a homocysteine residue.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the radical initiator is a photoinitiator selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, 1-hydroxy-cyclohexyl-phenyl-ketone and 2,2-dimethoxy-1,2-diphenylethan-1-one.

### Claim 5 (depends on 4)

The method according to claim 4, wherein a thiol-ene reaction is initiated by exposing the photoinitiator to a light having a wavelength and an intensity during an exposure time, wherein:
when the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP), the wavelength is approximately 360 nm, 372 nm, 380 nm, or 385 nm;
when the photoinitiator is 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, the wavelength is approximately 276 nm or 331 nm;
when the photoinitiator is 1-hydroxy-cyclohexyl-phenyl-ketone, the wavelength is approximately 246 nm, 280 nm, or 333 nm; and
when the photoinitiator is 2,2-dimethoxy-1,2-diphenylethan-1-one, the wavelength is approximately 254 nm or 337 nm.

### Claim 6 (depends on 4)

The method according to claim 4, wherein the method further comprises controlling the amount of the photoinitiator, the intensity of the light and the time the photoinitiator is exposed to the light.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the thiol-ene reaction reaches between about 70% completion and about 95% completion.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the ene compound is an ene-modified biocompatible monomer.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the ene compound is a norbornene-modified polyethylene glycol (PEG).

### Claim 10 (depends on 8)

The method according to claim 8, wherein the polypeptide further comprises a peptide sequence known to be sensitive to a protease.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the thiol-ene reaction provides a biocompatible cross-linked degradable hydrogel polymer.

### Claim 12 (depends on 1)

The method of claim 1, wherein the ene compound comprises one or more norbornene groups.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the polypeptide is selected from peptide or protein hormones, growth factors, cytokines, interleukins, receptors, solubilized receptors, therapeutic proteins, enzymes and structural proteins.