Process for the conjugation of a peptide or protein with a reagent comprising a leaving group including a portion of PEG
The invention relates to novel conjugating reagents capable of reaction with at least one nucleophile present in a peptide or protein, which contain at least one leaving group which is lost on reaction with said nucleophile, in which the leaving group includes a portion —(CH 2 CH 2 O) n —, in which n is a number of six or more; and novel processes for the preparation of conjugates containing peptides or proteins made using such reagents.
1. A conjugating reagent, wherein the conjugating reagent is of the formula (Ic), (Ic′), (Id), (Id′), (Ie), (Ie′), (IIa), (IIIa) or (IIIb):
wherein:
D represents a payload which is a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent;
Q represents a linking group;
W represents an electron-withdrawing group;
A represents a C 1-5 alkylene or alkenylene chain;
B represents a bond or a C 1-4 alkylene or alkenylene chain;
Ar represents an optionally substituted aryl;
R 4 represents a hydrogen atom or a C 1-4 alkyl group;
R 4′ represents a hydrogen atom;
p represents 0 or an integer of from 1 to 4;
each L independently represents a leaving group, at least one of which, and in cases where there are two, both include a —(CH 2 CH 2 O) n — in which n is a number of six or more; and
L′ is a leaving group which includes a —(CH 2 CH 2 O) n — in which n is a number of six or more or is a leaving group of another structure; or
the two Ls together, or the L and L′ together, represent a leaving group which includes a —(CH 2 CH 2 O) n1 — in which n1 is a number of two or more;
and wherein the portion —(CH 2 CH 2 O) n — has a molecular weight of up to 5 kDa.
2. A conjugating reagent as claimed in claim 1 , in which n is from 6 to 9.
3. A reagent as claimed in claim 1 , in which said leaving group is of the formula —SP, —OP, —SO 2 P, —OSO 2 P, —N + PR 2 R 3 , or is a group of formula —S—P—S—, —O—P—O—, —SO 2 —P—SO 2 —, —OSO 2 —P—OSO 2 —, or —N + R 2 R 3 —P—N + R 2 R 3 —, in which P is a group which includes a portion —(CH 2 CH 2 O) n — and each of R 2 and R 3 independently represents a hydrogen atom or a 4alkyl group or a group P.
4. A reagent as claimed in claim 3 , in which said leaving group is of the formula —SO 2 P.
5. A reagent as claimed in claim 1 , in which said leaving group includes —(CH 2 CH 2 O) n —R 1 where R 1 is a capping group; or in which said —(CH 2 CH 2 O) n — group has two points of attachment within the reagent.
6. A reagent as claimed in claim 1 , in which said leaving group is —S—(CH 2 CH 2 O) n —(CH 2 CH 2 )—S—, —O—(CH 2 CH 2 O) n —(CH 2 CH 2 )—O—, —SO 2 —(CH 2 CH 2 O) n —(CH 2 CH 2 )—SO 2 —, —OSO 2 —(CH 2 CH 2 O) n —(CH 2 CH 2 )—OSO 2 —, —N + R 2 R 3 —(CH 2 CH 2 O) n —(CH 2 CH 2 )—N + R 2 R 3 —, or one of the groups of formula
where R 1 is a capping group and each of R 2 and R 3 independently represents a hydrogen atom or a C 1-4 alkyl group.
7. A reagent as claimed in claim 6 , in which said leaving group is —SO 2 —(CH 2 CH 2 O) n —(CH 2 CH 2 )—SO 2 —.
8. A reagent as claimed in claim 5 , in which R 1 represents a hydrogen atom, a C 1-4 alkyl group, an optionally substituted aryl group, or a group of formula —CH 2 CH 2 CO 2 H or —CH 2 CH 2 NH 2 .
9. A reagent as claimed in claim 1 , in which D-Q includes a drug or a polymer or both a drug and a polymer.
10. A reagent as claimed in claim 9 , in which the drug if present is a cytotoxic drug, and the polymer if present is polyethylene glycol.
11. A conjugating reagent as claimed in claim 1 , in which the conjugating reagent is of formula (Ic).
12. A conjugating reagent as claimed in claim 1 , in which D is a cytotoxic agent or toxin.
13. A conjugating reagent as claimed in claim 1 , in which D is an auristatin or a maytansinoid.
14. A conjugating reagent as claimed in claim 1 , in which Q represents a linking group containing a degradable group containing a group susceptible to enzymatic degradation.
15. A conjugating reagent as claimed in claim 1 , in which Q represents a linking group containing a group of the formula:
wherein AA represents a protease-specific amino acid sequence.
16. A conjugating reagent as claimed in claim 15 , in which AA is Val-Cit.
17. A conjugating reagent as claimed in claim 1 , in which W is a keto group.
18. A conjugating reagent as claimed in claim 1 , in which A is —CH 2 — and B is a bond.
19. A conjugating reagent as claimed in claim 1 , in which Lisa group of the formula —SO 2 —(CH 2 CH 2 O) 7 —CH.
20. A process for the conjugation of a peptide or protein, which comprises reacting said peptide or protein with a conjugating reagent as claimed in claim 1 .
21. A process as claimed in claim 20 , in which the protein is a receptor or ligand binding protein or an antibody or antibody fragment.