Modified tyrosine amino acids, peptides containing them, and their use for detecting hydrolase enzyme activity

Method for the manufacturing of a double modified tyrosine monomer, comprising the steps of:

• (a) modifying tyrosine monomers with a first protecting group on the amine residue, thereby obtaining a modified tyrosine monomer according to formula VII;
• wherein X1 and/or X2 is selected from —N3, —NO2, —Br, or H, wherein either X1 or X2 is not H, and wherein P1 is a first protecting group;
• (b) modifying the modified tyrosine monomers obtained from step (a) by esterification of the carboxyl group, thereby obtaining a modified tyrosine monomer according to formula VIII;
• wherein P2 is a second protecting group;
• (c) modifying the modified tyrosine monomers obtained from step (b) by providing the remaining hydroxyl group with a functional group provided with secondary protecting groups, thereby obtaining a modified tyrosine monomer according to formula IX;
• wherein Y is chosen from —PO32− with y equal to 2 or 3 and esters thereof;
• and;
• wherein said Y group is provided with n P3 groups, wherein P3 is a third protecting group and n is an integer selected from 1 or 2.

A modified tyrosine monomer according to formula IX

• wherein P1, P2 and P3 are orthogonal protecting groups selected from phthalimido, nitrobenzenesulphonyl, fluorenylmethyloxycarbonyl (Fmoc), tert-Butyloxycarbonyl (Boc), allyloxycarbonyl (Aloc), carboxybenzyl (Cbz), allyl, C1 to C8 alkyl, benzyl, (2-phenyl-2-trimethylsilyl)ethyl (PTMSE), phenyl, succinimidyl, pentafluorophenyl, pentachlorophenyl, 4-nitrophenyl, t-butyl and 2-cyanoethyl;
• wherein X1 and/or X2 is selected from —N3, —NO2, or H, wherein either X1 or X2 is not H;
• wherein Y is chosen from —PO32−with y equal to 2 or 3 and esters thereof;
• and
• wherein said Y group is provided with n P3 groups, wherein P3 is a third protecting group and n is an integer selected from 1 or 2.