# US 8,785,595 B2 · Functionalising reagents and their uses

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- Number: US 8,785,595 B2
- Document ID: US8785595B2
- Status: Granted
- Kind: B2
- Application number: 13/140,690
- Filed: 2009-12-21
- Published: 2012-04-26
- Granted: 2014-07-22
- Earliest claimed: 2008-12-19
- Assignees: Glythera Limited
- Applicants: Andrew Graham Watts; Terrence Kantner; Amanda Barbara MacKenzie
- Inventors: Andrew Graham Watts; Terrence Kantner; Amanda Barbara MacKenzie
- Web page: https://patentgrep.com/patent/US8785595B2

## CPC

- C07D 239/28: with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07D 213/30: Oxygen atoms
- C07D 213/38: having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
- C07D 213/46: Oxygen atoms
- C07D 213/55: Acids; Esters
- C07D 213/80: in position 3
- C07H 15/26: Acyclic or carbocyclic radicals, substituted by hetero rings
- C07K 1/13: Labelling of peptides
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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)

## Abstract

Reagents and methods for functionalizing polypeptides with moieties poly (alkylene glycol) molecules and glycan groups are disclosed that are based on a functionalizing reagent which comprises a nitrogen containing heterocyclic aromatic ring having a vinyl substituent that is capable of reacting with one or more thiol groups that are naturally present, or have been introduced into, the polypeptide, for example by employing a thiol group of one or more cysteine residues. The functionalizing reagent is covalently linked to a poly (alkylene glycol) molecule, such as a polyethylene glycol (PEG) molecule, or a glycan group so that the reaction between the vinyl group and the thiol group in the polypeptide covalently links the polypeptide to the poly (alkylene glycol) molecule and/or the glycan group.

## Claims

### Claim 1

A method for modifying a polypeptide having at least one reactive thiol group, the method comprising contacting the polypeptide with a functionalising reagent which comprises a nitrogen containing heterocyclic aromatic ring having a vinyl substituent capable of reacting with the at least one thiol group of the polypeptide, wherein the functionalising reagent is covalently linked to a poly (alkylene glycol) molecule or a glycan group, so that the vinyl substituent of the functionalising reagent reacts with the thiol group of the polypeptide, thereby to covalently link the poly (alkylene glycol) molecule or glycan group to the polypeptide through functionalising reagent.

### Claim 2 (depends on 1)

The method of claim 1, further comprising the initial step of reacting a precursor functionalising reagent comprising a nitrogen containing heterocyclic aromatic ring having a vinyl substituent with the poly (alkylene glycol) molecule or glycan group to produce the functionalising reagent.

### Claim 3 (depends on 1)

The method of claim 1, wherein the poly (alkylene glycol) molecule is a polyethylene glycol (PEG) molecule.

### Claim 4 (depends on 1)

The method of claim 1, wherein the nitrogen containing heterocyclic aromatic ring is a pyridine, pyrimidine or azidine ring.

### Claim 5 (depends on 1)

The method of claim 1, further comprising determining the location of said at least one reactive thiol group in the polypeptide, and optionally protecting said reactive thiol group(s).

### Claim 6 (depends on 1)

The method of claim 1, further comprising the initial step of modifying a parent polypeptide by chemical reaction or by site directed mutagenesis to produce a variant polypeptide comprising a thiol group at one or more desired positions of the polypeptide.

### Claim 7 (depends on 1)

The method of claim 1, wherein the thiol group is present in a cysteine residue.

### Claim 8 (depends on 1)

The method of claim 1, wherein the functionalising reagent which comprises a nitrogen containing heterocyclic aromatic ring having a vinyl substituent is represented by the general formula:
wherein:
X and Y are independently selected from CH or N;
or R3 is selected from hydrogen or a substituent selected from halo, hydroxy, ether, formyl, acyl, carboxy, ester, acyloxy, amido, acylamido, thioamido, tetrazolyl, amino, nitro, nitroso, azido, cyano isocyano, cyanato, isocyanato, thiocyano, isothiocyano, thioether, sulfonic acid, sulfonate, sulfone, sulfonyloxy, sulfinyloxy, sulfamino, sulfonamino, sulfinamino, sulfamyl, sulfonamido, C1-7 alkyl.

### Claim 9 (depends on 8)

The method of claim 8, wherein X and Y are both CH or one of X and Y is CH and the other is N.

### Claim 10 (depends on 8)

The method of claim 8, wherein one of R1 or R2 comprises a poly (alkylene glycol) molecule or a glycan group.

### Claim 11 (depends on 8)

The method of claim 8, wherein both of R1 and R2 comprise a poly (alkylene glycol) molecule and/or a glycan group.

### Claim 12 (depends on 8)

The method of claim 8, wherein one of R1 and R2 comprises a poly (alkylene glycol) molecule and the other of R1 and R2 comprises a glycan group.

### Claim 13 (depends on 8)

The method of claim 8, wherein the R1 and/or R2 groups for linking the poly (alkylene glycol) molecule and/or the glycan group to the nitrogen containing heterocyclic aromatic ring are independently selected from —(CH2)n—Z—(CH2)o—R or —C(O)—Z—(CH2)o—R.

### Claim 14 (depends on 8)

The method of claim 8, wherein in the R1 and R2 groups, o is zero and the groups may be represented as —(CH2)n—Z—R or —C(O)—Z—R.

### Claim 15 (depends on 8)

The method of claim 8, wherein, the functionalising reagent is represented by one of the general formulae:
wherein at least R1, and optionally R2, are covalently linked to a poly (alkylene glycol) molecule and/or a glycan group;
wherein
R1, and optionally R2, are independently selected from:
—(CH2)n—Z—(CH2)o—C(O)—R, where Z is O, S or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—(CH2)n—Z—(CH2)o—R, where Z is O, S or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—C(O)—Z—(CH2)o—R, Z is O, S or NH, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—(CH2)n—Z—C(O)—(CH2)o—R, where Z is O, S or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—Y—(CH2)n—Z—C(O)— (CH2)o—R, where Y is an aryl group, O, S or NH, Z is O or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—Y—R, where Y is O or S, R is a hydrogen, a glycan, a poly (alkylene glycol) molecule, aryl or C1-10 alkyl;
with the proviso that when R2 is not linked to a poly (alkylene glycol) molecule or a glycan group, it may additionally be selected from hydrogen or an electron withdrawing group, such as halogen (F, Cl or Br), —NO2, —CO2H, —CO2R4, COR4, —CHO, —CN, —CF3, —SO2NR4R5, alkyl or phenyl, where R4 and R5 are independently selected from hydrogen or C1-10 alkyl;
or R2 and R3 together form a fused (hetero) aromatic ring substituent which may include, but is not limited to, an indole, indazole, benzimidazole, quinoline, isoquinoline, aziradine or a purine;
wherein:
at least R1, and optionally R2, are covalently linked to a poly (alkylene glycol) molecule and/or a glycan group;
and further wherein:
R1, and optionally R2, are independently selected from:
—(CH2)n—Z—(CH2)o—C(O)—R, where Z is O, S or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—(CH2)n—Z—(CH2)n—R, where Z is O, S or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—C(O)—Z—(CH2)o—R, Z is O, S or NH, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—(CH2)n—Z—C(O)—(CH2)o—R, where Z is O, S or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group; or
—Y— (CH2)n—Z—C(O)—(CH2)o—R, where Y is an aryl group, O, S or NH, Z is O or NH, n is 0 to 10, o is 0 to 10 and where R is a hydrogen, a poly (alkylene glycol) molecule or a glycan group, or —Y—R, where Y is O or S, R is a hydrogen, a glycan, a poly (alkylene glycol) molecule, aryl or C1-10 alkyl;
with the proviso that when R2 is not linked to a poly (alkylene glycol) molecule or a glycan group, it may additionally be selected from hydrogen or an electron withdrawing group, such as halogen (F, Cl, or Br), —NO2, —CO2H, —CO2R4, COR4, —CHO, —CN, —CF3, —SO2NR4R5, alkyl or phenyl, where R4 and R5 are independently selected from hydrogen or C1-10 alkyl.

### Claim 16 (depends on 1)

The method of claim 1, wherein the glycan is a carbohydrate group for glycosylating the polypeptide.

### Claim 17 (depends on 16)

The method of claim 16, wherein the carbohydrate group is a naturally occurring or synthetic monosaccharide, oligosaccharide or polysaccharide.

### Claim 18 (depends on 16)

The method of claim 16, wherein the glycan is a N-linked or 0-linked saccharide group.

### Claim 19 (depends on 1)

The method of claim 1, wherein the poly (alkylene glycol) molecule group has a molecular weight between IkDa and 5 OkDa.

### Claim 20 (depends on 1)

The method of claim 1, wherein the poly (alkylene glycol) molecule or glycan group is conjugated to the polypeptide to modify a pharmacological property of the polypeptide.

### Claim 21 (depends on 20)

The method of claim 20, wherein the pharmacological property is stability, biological half-life, water solubility and/or immunologic characteristics of the polypeptide.

### Claim 22 (depends on 1)

The method of claim 1, wherein the polypeptide is an erythropoietin, an interferon, an interleukin, a chemokine, a lymphokine, a cytokine, insulin, a monoclonal antibody or fragment thereof, a recombinant antibody or fragment thereof, a blood-clotting factor, a colony-stimulating factor, a growth hormone, a plasminogen activator, a virally-derived peptide, a reproductive hormone, a therapeutic enzyme or a carrier protein for a conjugate vaccine.

### Claim 23 (depends on 22)

The method of claim 22, wherein the conjugate vaccine is formed between a carrier protein and one or more glycan groups which are antigenic polysaccharides.

### Claim 24

A method which comprises contacting a polypeptide having at least one reactive thiol group with a functionalising reagent which comprises a nitrogen containing heterocyclic aromatic ring having a vinyl substituent capable of reacting with the at least one thiol group of the polypeptide, wherein the functionalising reagent is covalently linked to a poly (alkylene glycol) molecule or a glycan group, so that the vinyl substituent of the functionalising reagent reacts with the thiol group of the polypeptide, thereby to covalently link the poly (alkylene glycol) molecule or glycan group to the polypeptide through functionalising reagent.