Catalytic Antibody – AZ-28 Oxy Cope Rearrangement Biocatalyst

1. Catalytic Antibody – AZ-28 Oxy Cope Rearrangement Biocatalyst CHEM*4450 – Biochemistry and Structure of Macromolecules Seminar Series Presented By: Woo-Jin Yoo

2. Seminar Outline • Introduction • Implications For Transition-State Analogs And Catalytic • Antibodies • AZ-28: Oxy-Cope Rearrangement Catalytic Antibody • Structure And Function Relationship Of AZ-28 With • Transition State Analog • Structure And Function Relationship Of AZ-28 And • Germline Antibody • Concluding Remarks • References

3. Introduction Rationale for the development of biocatalyst • high regio- and stereoselectivity • environmentally friendly • non-toxic Advantages: Biocatalysts: enzymes, bioengineered microorganisms, ribozymes, catalytic antibodies Advantage of catalytic antibodies: Potential to design a biocatalyst for a specific reaction

4. Implications For Transition-State Analogs And Catalytic Antibodies How do enzymes accelerate chemical reactions? For one substrate to product situation:

5. Corresponding Energy Diagram: Enzymes accelerate chemical reactions by stabilizing the transition state.

6. What are the available physical/chemical forces available for binding and catalysis? • Van der Waal • Hydrogen bonding • Hydrophobic effect • Ionic Interactions

7. Designing a protein to catalyze a chemical reaction: Idea: Proteins which binds transition state strongly should be able to catalyze a chemical reaction Application: Immunize an animal using a transition state analog as a hapten to form antibodies that bind to the transition state analog

8. Example: Oxy-Cope rearrangement catalytic antibody AZ-28

9. 1. Determination and synthesis of the hapten (transition state analog) 2. Attachment of hapten to a macromolecule BSA = Bovine Serum Albumin KLH = Keyhole Limpet Hemocyanin

10. 3. Preparation of monoclonal antibodies

11. AZ-28: Oxy-Cope Rearrangement Catalytic Antibody Background Information • rearrangement is based on a diradical, cyclohexane TS intermediate • driven by the formation of a keto-enol compound

12. Why is this reaction slow/not possible at room temperature? • transition state is a six-membered ring in the chair conformation • in solution, there are possible rotation of sigma bonds

13. Hapten design: Rationale • transition state is a cyclohexane intermediate • strong preference for aromatic rings for catalytic antibodies • CONH(CH2)3COOH – is the tether to BSA/KLH

14. Structure And Function Relationship Of AZ-28 With Transition State Analog AZ-28: Unliganded MatureOxy- Cope Catalytic Antibody

15. Binding interactions between AZ-28 and Transition State Analog • 5-phenyl group • at the bottom of cavity • surrounded by large number of • aromatic and hydrophobic residues • -stacking with H103His • 2-phenyl group • at the opening of the binding pocket • orientation is fixed by -stacking with • H96His and van der waal interaction • with side chain of L91Tyr • Cyclohexane ring • position fixed by H-bonding with OH • group and the imidazole ring of H96His • van der waal contact with L33Asn • H101Asp

16. Mechanistic proposal for the Oxy-Cope rearrangement with AZ-28 1. Entopic Effect Extended conformation is fixed into the energetically unfavorable conformation by the binding site of the antibody G = H - TS Fixing conformation = S , G 

17. 2. Electronic Effect Side chain of H96His and H-bonding of bridging water to H50Glu increases the electron density of oxygen Increased electron density on the oxygen will increase the rate of Oxy-Cope rearrangement

18. Structure And Function Relationship Of AZ-28 And Germline Antibody Fab of Mature Antibody Fab of Germline Antibody

19. What the heck is going on? AZ-28 binds the TS analog more tightly than the germline antibody, but the germline antibody is a better catalyst

20. Comparison of AZ-28 with germline antibody AZ-28 bound with TSA germline bound with TSA

21. Reason for increased catalysis of germline antibody Recall: The transition state of the Oxy-Cope rearrangement is a diradical The radical can be stabilized by the aromatic group

22. Molecular orbital reasons for increased catalytic activity Note: stabilization of the transition state decreases the energy requirements for catalysis When radical is in the same plane as aromatic ring When radical is perpendicular to the aromatic ring • The germline antibody fixes the TSA so that the aromatic rings are • 63.2o (5-phenyl) and 57.9o (2-phenyl) to the cyclohexane framework • AZ-28 fixes the TSA so that the aromatic rings are 81o (5-phenyl) • and 85o (2-phenyl) to the cyclohexane framework

23. Electron density diagram of the active site for AZ-28

24. Structural reasons for difference in aromatic ring angle between AZ-28 and germline antibody Primary sequence difference

25. Structural basis for catalytic properties of germline and affinity matured antibody Only L34 amino acid residue is at the active site AZ-28 – L34Asn Germline – L34Ser Liganded AZ-28 – 2.6 Å Unliganded AZ-28 – 3.2 Å Liganded Germline – 3.0 Å Unliganded Germline – 3.7 Å Result: Increased flexibility of active site for germline antibody lowers the rotational barrier for the C2-phenyl

26. Concluding Remarks Comments: AZ-28 binds more tightly to the TSA than germline antibody. However, germline antibody is a better catalyst Reason: Flaw in the design of TSA. True TS possess sp2 carbons attached to the aromatic groups. TSA have sp3 carbons and in solution, the aromatic groups prefer to be perpendicular to the cyclohexane framework

27. References • Ulrich, H.D., Mundorff, E.C., Santarsiero, B.D., Driggers, E.M., Stevens, R.C., and • Schultz, P.G. (1997) Nature 389, 271-275. • Driggers, E.M., Cho, H.S., Liu, C.W., Katzka, C.P., Braisted, A.C., Ulrich, H.D., • Wemmer, D.E. and Schultz, P.G. (1998) J. Am. Chem. Soc., 120, 1945-1958. • Mundorff, E.C., Hanson, M.A., Varvak, A., Ulrich, H.D., Schultz, P.G., and Stevens, • R.C. (2000) Biochemistry39, 627-632. • Braisted, A.C. and Schultz, P.G. (1994) J. Am. Chem. Soc.,116, 2211-2212. • Mader, M.M., and Bartlett, P.A. (1997) Chem. Rev., 97, 1281-1301.