Conjugated biological molecules and their preparation

1. A process for the preparation of a protein-polymer conjugate of formula I,

wherein

one of X and X′ is a polymer and the other is a hydrogen atom;

Q is a linking group;

W is selected from the group consisting of a keto group, an ester group, a sulphone group, a reduced keto group, a reduced ester group, and a reduced sulphone group;

and if X represents a polymer, X′ and W together with the interjacent atoms may form a ring;

A is a C 1-5 alkylene or alkenylene chain;

B is a bond or a C 1-4 alkylene or alkenylene chain; and

Z is a single protein linked to A and B via two thiol groups generated by the reduction of a disulfide bridge in the protein;

said method comprising the steps of:

(a) reducing a disulfide bridge in the protein; and

(b) reacting the reduced protein with either

(i) a compound of formula II

wherein X, X′, Q, A and B are defined as in formula I;

W′ is selected from the group consisting of a keto group, an ester group, and a sulphone group, and if X represents a polymer, X′ and W′ together with the interjacent atoms may form a ring;

and

L is independently a leaving group; or

(ii) a compound of formula III

wherein X, X′, Q and A are defined as in formula I,

W′ is selected from the group consisting of a keto group, an ester group, and a sulphone group, and if X represents a polymer, X′ and W′ together with the interjacent atoms may form a ring;

L is a leaving group; and m is an integer from 1 to 4.

2. The process according to claim 1 , in which each leaving group L is independently selected from the group consisting of SR, —SO 2 R, —OSO 2 R, —N + R 3 , —N + HR 2 , —N + H 2 R, halogen, and —OØ, in which R represents an alkyl or aryl group and Ø represents a substituted aryl group containing at least one electron withdrawing substituent.

3. A compound of the general formula II

wherein one of X and X′ is a polymer and the other is a hydrogen atom;

Q is a linking group;

W′ is selected from the group consisting of a keto group, an ester group, and a sulphone group, and if X represents a polymer, X′ and W′ together with the interjacent atoms may form a ring;

A is a C 1-5 alkylene or alkenylene chain;

B is a bond or a C 1-4 alkylene or alkenylene chain; and

L is independently a leaving group.

4. A compound of the general formula III

wherein one of X and X′ is a polymer and the other is a hydrogen atom;

Q is a linking group;

W′ is selected from the group consisting of a keto group, an ester group, and a sulphone group; and if X represents a polymer, X′ and W′ together with the interjacent atoms may form a ring;

A is a C 1-5 alkylene or alkenylene chain;

L is a leaving group;

and m is an integer from 1 to 4.

5. A polymer conjugating reagent comprising a compound of claim 3 or 4 in an aqueous reaction medium.

6. The process according to claim 1 further comprising reducing W′ to produce a protein-polymer conjugate of formula I, wherein W is selected from the group consisting of a reduced keto group, a reduced ester group, and a reduced sulphone group.

7. The process according to claim 1 , wherein polymer X or X′ is a homo- or copolymer selected from the group consisting of polyalkylene glycols, polyvinylpyrrolidones, polyacrylates, polymethacrylates, polyoxazolines, polyvinylalcohols, polyacrylamides, polymethacrylamides, HPMA copolymers, polyesters, polyacetals, poly(ortho ester)s, polycarbonates, poly(imino carbonate)s, polyamides, copolymers of divinylether-maleic anhydride and styrene-maleic anhydride, polysaccharides, and polyglutamic acids.

8. The process according to claim 1 , wherein polymer X or X′ is a polyethylene glycol.

9. The process according to claim 1 , wherein linking group Q is independently selected from the group consisting of a direct bond, an alkylene group, and an optionally-substituted aryl or heteroaryl group, any of which may be terminated or interrupted by one or more oxygen atoms, sulphur atoms, keto groups, —O—CO— groups and/or —CO—O— groups, or —NR groups in which R represents an alkyl or aryl group.

10. The process according to claim 1 , which is carried out in an aqueous medium.

11. The compound of claim 3 , wherein each L is independently selected from the group consisting of SR, —SO 2 R, —OSO 2 R, —N + R 3 , —N + HR 2 , —N + N 2 R, halogen, and —OØ, in which R represents an alkyl or aryl group and Ø represents a substituted aryl group containing at least one electron withdrawing substituent.

12. The compound of claim 3 , wherein Q is independently selected from the group consisting of a direct bond, an alkylene group, and an optionally-substituted aryl or heteroaryl group, any of which may be terminated or interrupted by one or more oxygen atoms, sulphur atoms, keto groups, —O—CO— groups and/or —CO—O— groups, or —NR groups in which R represents an alkyl or aryl group.

13. The compound of claim 3 , wherein polymer X or X′ is a polyethylene glycol.

14. The compound of claim 4 , wherein L is selected from the group consisting of SR, —SO 2 R, —OSO 2 R, —N + R 3 , —N + HR 2 , —N + H 2 R, halogen, and —OØ, in which R represents an alkyl or aryl group and Ø represents a substituted aryl group containing at least one electron withdrawing substituent.

15. The compound of claim 4 , wherein Q is independently selected from the group consisting of a direct bond, an alkylene group, and an optionally-substituted aryl or heteroaryl group, any of which may be terminated or interrupted by one or more oxygen atoms, sulphur atoms, keto groups, —O—CO— groups and/or —CO—O— groups, or —NR groups in which R represents an alkyl or aryl group.

16. The compound of claim 4 , wherein polymer X or X′ is a polyethylene glycol.