The invention relates to substrates for O<6>-alkylguanine-DNA alkyltransferases (AGT) of formula R1-A-X-CH2-R3-R4-L1, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R1 is a group -R2-L2 or a group R5, R2 and R4 are, independently of each other, a linker, R3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH2, R5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L1 is a label, a plurality of same or different labels, a bond connecting R4 to A forming a cyclic substrate , or a further group -R3 CH2-X-A-R1, and L2 is a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O<6->