Site-specific, covalent bioconjugation of proteins
Heterofunctional crosslinking groups are provided having the formula: wherein W is a covalent core component; L 1 , L 2 and L 3 are independently linking groups; X is a non-covalent or reversibly covalent protein tag binder; Y is a activatable covalent linking group; and Z is a protected or unprotected covalent crosslinking group.
1. A heterofunctional crosslinking reagent having the formula:
wherein
L 1 and L 2 are each independently selected from the group consisting of a bond, a substituted or unsubstituted (C 2 –C 24 ) alkylene group, a substituted or unsubstituted (C 2 –C 24 ) heteroallcylene group, a polyethyleneglycol group, a polyalcohol group, a polyamine group, a poiyester group and a polyphosphodiester group;
-L 3 -Z is an optionally protected amino acid side chain having a pendant reactive group, wherein said amino acid is selected from the group consisting of lysine, cysteine, serine, aspartic acid, glutamic acid, and threonine;
X is a non-covalent protein tag binder that specifically binds to a protein tag portion of a protein; and
Y is a photoactivatable covalent crosslinking group adapted to covalently link the heterofunctional crosslinking reagent at or adjacent to said protein tag, said photoactivatable covalent crosslinking group is a member selected from the group consisting of aryl ketones, azides, diazo compounds, diazirenes, and ketenes.
2. A heteroflinctional crosslinking reagent of claim 1 , wherein L 1 is a cleavable linking group.
3. A heterofunctional crosslinking reagent of claim 1 , wherein X is selected from the group consisting of metal chelating groups, peptides, an organoarsenical moiety and small molecule ligands or inhibitors.
4. A protein conjugate comprising a protein and a heterofunctional crosslinking reagent, said conjugate having the formula:
wherein
L 1 and L 2 are each independently selected from the group consisting of a bond, a substituted or unsubstituted (C 2 –C 24 ) alkylene group, a substituted or unsubstituted (C 2 –C 24 ) heteroalkylene group, a polyethyleneglycol group, a polyalcohol group, a polyamine group, a polyester group and a polyphosphodiester group;
-L 3 -Z is an optionally protected amino acid side chain having a pendant reactive group, wherein said amino acid is selected from the group consisting of lysine, cysteine, serine, aspartic acid, glutamic acid, and threonine;
X is a non-covalent protein tag binder that specifically binds to a protein tag portion of said protein; and
Y′ is the residue of a photoactivatable covalent crosslinking group after formation of a covalent linkage to said protein, said photoactivatable covalent crosslinking group covalently attached at or adjacent to said protein tag portion of said protein, said photoactivatable covalent crosslinking group is a member selected from the group consisting of aryl ketones, azides, diazo compounds, diazirenes, and ketenes.
5. A heterofunctional crosslinking reagent of claim 1 , wherein X is an antibody or antibody fragment.