This research program represents a collaboration between Professor Hirschmann, Smith and Christianson at the University of Pennsylvania and Professor Benkovic at the Pennsylvania State University (Section 5). The principal goal of the program is to produce catalytic antibodies which will promote efficient condensation of peptide fragments. The use of such antibodies to effect peptide bond formation should manifest the advantages of enzymatic peptide synthesis (i.e., minimal need for protecting groups, chemospecificity, and stereospecificity) and avoid the limitations associated with enzymes instability, enzyme-induced hydrolysis, and substrate related solubility problems. Importantly, one will not be restricted by the repertoire of available enzymes. These considerations lead us to propose the following specific aims for the 04-07 years of GM-45611: (1) To explore strategies for in situ activation of stable acyl fragments immediately prior to antibody-catalyzed couplings. To this end, the investigators will develop second-generation haptens that mimic tetrapeptides (i.e., transition limitations, and mechanism of catalysis by antibody 16G3 via: (a) studying a variety of substrates; (b) defining the kinetics of catalysis; and (c) determining the three-dimensional structures of Fab 16G3, both uncomplexed and complexed with a transition state analog and with substrates. (3) To develop antibodies capable of catalyzing peptide formation from inactivated acyl donors and larger fragments, the investigators will perform site-specific mutagenesis on antibody 16G3. (4) To prepare large amounts of 16G3 and its single-chain analog, as required for subgoals 2 and 3. They have already begun to generate 16G3 on a large scale (ca. 1-34g) at the Penn Cell Center. (5) In addition, the investigators propose to: (a) prepare FAb (or Fab') fragments of 16G3 by routine proteolysis; and (b) convert the hybridoma-cell RNA to single-chain antibodies (SCA). (6) To exploit the newly discovered phosphonyl trialkylammonium salts, refining and further developing our effective protocol for the synthesis of phosphonate esters and phosphonamides. Assuming that the concept of using catalytic antibodies in fragment condensation is viable, and that it can be applied to the coupling of longer fragments, condensation is, a suitable leap of faith would be to attempt to use a catalytic antibody to couple an appropriately C-terminally derovatozed S-peptide to the corresponding S-protein, both readily available from ribonuclease A. The reconstruction of ribonuclease A from its subtilisin degradation products would be a dramatic demonstration of the scope of this approach. This has previously been achieved enzymatically. The investigators will then apply catalytic antibodies to couple fragments of other known proteins and subsequently of peptides and proteins that incorporate uncoded amino acids.
| Savinov, Sergey N; Hirschmann, Ralph; Benkovic, Stephen J et al. (2003) Investigation of an antibody-ligase. Evidence for strain-induced catalysis. Bioorg Med Chem Lett 13:1321-4 |
| Smithrud, D B; Benkovic, P A; Benkovic, S J et al. (2000) Cyclic peptide formation catalyzed by an antibody ligase. Proc Natl Acad Sci U S A 97:1953-8 |
| Hirschmann, R; Smith 3rd, A B; Taylor, C M et al. (1994) Peptide synthesis catalyzed by an antibody containing a binding site for variable amino acids. Science 265:234-7 |
