The control of reactivity of functional groups on the side chains of amino acids and peptides is a potentially attractive field for applications of catalytic antibodies. The selective modification of amino acid side chains in peptides and proteins is important for many areas of chemistry, from organic synthesis to the irreversible inhibition of enzymes and the improvement of pharmacokinetic properties of peptide drugs.a However, the high level of control over chemo- and regioselectivity which is required for these transformations is not easily obtained by chemical or enzymatic approaches.b

Myristoylation of insulin at a lysine residue largely improves the bioavailability of the hormone.c

Esterase antibody 522C2 is the first catalytic antibody specifically designed to hydrolyze an ester group on the tyrosine phenolic side chain.d

The phenylphosponate of N-Cbz-Tyrosine was used as the transition state analogue. The carboxylate group of tyrosine was used to conjugate directly the phosphonate to cationised Bovine Serum Albumin (cBSA). We reasoned that holding the hapten in close contact with the carrier protein surface would result in a relatively "open" antigen binding site, able to recognize and hydrolize the substrate also when tyrosine is part of a peptide chain. For this reason the use of a linker between the hapten and the carrier was avoided.

Antibody 522c2 catalyzes the hydrolysis of N-Cbz-tyrosine bensoate 1 by a factor of 10⁴ and its activity is  highly specific for the S-enantiomer of tyrosine benzoate, mirroring the configuration of the hapten. While displaying such a high enantiospecificity, antibody 522C2 is able to hydrolyze a number of simplified esters 2 in which tyrosine is replaced by p-nitrophenol. the activity of 522C2 was tested also on two different tyrosine containing dipeptides: N-Cbz-TyrGlyOMe benzoate 3 and PheTyrOMe benzoate 4.

522c2 has been obtained also as a ScFv by the group of Prof. Carlo Pucillo at the University of Udine. An homolgy model of the fragment was built up by Dr. Neil Thomas and Simon Cross at the University of Nottingham.

As a consequence of the way chosen in the prepaparation of the immunogenic conjugate, the hapten in 522c2 (right) is placed in a very external position, if compared to the binding sites of other anti-phosphonate catalytic antibodies (left e)

The hapten interacts mainly with the heavy chain, two residues of arginin (H50, H52) and a tyrosine (L96) are in close contact with the phosphonate moiety of the transition state analogue.

a S.P. Vyas, P. Venugopalan, A. Sood and N. Mysore, Pharmazie 1997, 52, 339; D.K. Clodfelter, A.H. Pekar, D.M. Rebhun, K. A. Destrampe, H. A. Havel, S. R. Myers and M. L. Brader, Pharm. Res., 1998, 15, 254; M. Baudys, D. Letourneur, F. Liu, D. Mix, J. Jozefonvicz and S. W. Kim Bioconjugate Chem., 1998, 9, 176.

b B. T. Miller, Biochem. Biophys. Res. Commun. 1996, 218, 377; R. D. Lundbald, Techniques in Protein Modification, CRC Press, Boca Raton, 1995; A. W. Yem, H. A. Zurcher-Neely, K. A. Richard, N.D. Staite, R. L. Heinrikson and M. R. Deibel, J. Biol. Chem. 1989, 264, 17691; L. Gardossi, D. Bianchi and A. Klibanov, J. Am. Chem. Soc. 1991, 113, 6328.

c J. L. Wittingham, S. Havelund, I. Jonassen, Biochemistry, 1997, 36, 2826.

d F. Benedetti, F. Berti, A. Colombatti, M. Flego, L. Gardossi, P. Linda, S. Peressini, ChemComm, 2001, 8, 715.