Method of conjugating a polypeptide

A method for preparing a polypeptide conjugated to a payload comprising the step of:

• a) performing a Michael addition reaction with the maleimide entity in the molecule of formula (I):
• and a polypeptide molecule comprising at least one thiol group wherein:
• n is 0 or 1;
• m is 0 or 1;
• q is 0 or an integer in the range 1 to 12, for example 1 to 6, such as 2 or 3;
• Q is a bond or a residue from a conjugation component;
• X is C0-18 alkyleneC6-36 Aryl(-CR2═CH—)p, C0-18 alkyleneC6-36 aryl-CH2—CR2═CH—, C0-18 alkylene 5-36 memberedHeteroaryl(-CR═CH—)p, C0-18 alkylene 5-36 memberedHeteroaryl-CH2CR2═CH—, C0-18 Alkylene-CR2═CH—, C0-18 Alkylene-C≡C—,
• wherein the aryl or heteroaryl has 0, 1, 2, 3 or 4 substituent independently selected from the group comprising halogen, hydroxyl, C1-6 alkyl, C1-6 alkoxy, —COOR3, —COR3, —CN, —CF3, —NO2, —SO2, —SO3, —NR4R5, —PO4, and —(OCH2CH2)q—OR3;
• Y is oxo;
• Z is a saturated or unsaturated branched or unbranched C1-30 alkylene chain, wherein one or more carbons (such as 1, 2, 3, 4, 5, 6, 7 or 8) are optionally independently replaced by —O—, N and the chain is optionally bears one or more (such as 1, 2, 3 or 4) oxo substituents;
• R1 is H, a solid surface or a payload molecule;
• R2 is a substituent, for example selected from H, halogen, hydroxyl, —C1-6 alkyl, —C1-6 alkoxy, —COOR3, —COR3, —CN, —CF3, —NO2, —SO2, —SO3, —NR4R5, —PO4, C6-10 ArylC0-6 alkylene-, C6-10 HeteroarylC0-6 alkylene-;
• R3 is H or C1-6 alkyl;
• R4 is H or C1-6 alkyl;
• R5 is H or C1-6 alkyl;
• b) wherein R1 is a payload molecule or a solid surface hydrolysing the resultant thio-succinimide entity formed by the reaction of compound of formula (I) and the polypeptide, or
• c) wherein R1 is H performing a conjugation with a payload a conjugation component or solid surface followed by hydrolysing the t thio-succinimide entity formed by the reaction of compound of formula (I) and the polypeptide.