1. Insights into the Mechanism of Michael A. Cole Scripps-Oxford Laboratory 16 May 2007 Native Chemical Ligation S H2NH2N

2. Overview of Talk Background Questions to Answer Theory Basis for Study Progress Future Work Bruce Merrifield Nobelprize.org

3. Solid Phase Peptide Synthesis Why chemical synthesis? Before expression developed as method to gain access to proteins Specific control over side chain modifications SPPS developed by Bruce Merrifield in 1962 On resin allows for easy handling, higher purity, higher quantities (better solubility), and automation

4. Native Chemical Ligation Developed as a method for coupling two unprotected peptides in aqueous solution to form a native chemical bond The reaction of a c- terminal thioester with an n-terminal cysteine, generally in 6 M Gn·HCl pH 7 Developed to provide synthetic access to proteins

5. Questions to Answer Is cysteine the only moiety you can use on the N-terminus? Is a thioester the only activating group you can use on the C-terminus? Does the reaction proceed through a thioester intermediate?

6. Questions to Answer VS

7. Questions to Answer Why not?

8. Nucleophilic Reactivity Analyze the electrophile and nucleophile Optimize the reactivity of both mercaptopropionic acid leucine N-terminus/nucleophileC-terminus/electrophile

9. Reactivity of C-terminus nucleophilic acyl substitutions

10. C-terminus Jencks et al. J. Biol. Chem. (1960) 235, 3608-3614 A Comparison of Free Energy of Hydrolysis Thioesters 7,700 cal/mol Oxygen esters 5,100 cal/mol Peptide bond 500 cal/mol

11. Effect of pH on the reaction rate

12. pH 7 majority is HS-R-NH 3 + pH 8 70% HS-R-NH 3 +, 20% - S-R-NH 3 +, 10% HS-R-NH 2 - S-R-NH 3 + always greater than HS-R-NH 2

13. Effect of pH on the reaction rate Jencks, WP. Catalysis and Chemistry in Enzymology (1969)

14. Insights into the Mechanism Studied the effect of aromatic thiol catalysts on the rate of the NCL The mechanistic conclusion was that the transthioesterification is the rate limiting portion of the reaction Johnson and Kent. J. AM. CHEM. SOC. (2006) 128, 6640-6646

15. Insights into the Mechanism Originally thought thiols with lower pKas would be better catalysts Experiments revealed that the opposite was the case Thiol-thioester exchange reaction occurs faster with thiols with higher pKas (better nucleophiles) Johnson and Kent. J. AM. CHEM. SOC. (2006) 128, 6640-6646

16. Insights into the Mechanism Johnson and Kent. J. AM. CHEM. SOC. (2006) 128, 6640-6646 Fraction ligated vs pKa in transthioesterifications

17. Project Outline Synthesis of a library of modified C and N terminal peptides Synthesis of LYRAL C-terminal arylesters and MPAL thioalkyl ester Synthesis of N-terminal penicillamine, isocysteine, S or C - LYAA-NH2 Various phenols pKa 7.14-10 Measure rate constants of the NCL between the C-terminal peptide esters and thioesters and N-terminal peptides by HPLC Measure the pKas of the N-terminal amino acid side-chain and primary amine group by 1 H NMR Determine kinetics of various reaction conditions pH, buffer composition and concentration

18. C-terminus modifications LYRAL-MPAL LYRAL LYRALCLYLAA

19. N-terminus modifications CLYLAA SLYLAA Penicillamine-LYLAA Homocysteine-LYLAA

25. Example of Results HPLC traces

26. Example of Results Mass Spectrum m/z expected 634.8; found 635.0m/z expected 710.9; found 711.5

27. Future Work Determine initial rates of reaction pKa determination of N-terminus derivatives Reaction optimization Expanding to serine (changing conditions) Observe reaction intermediates with 13 C NMR

28. Questions to Answer Is cysteine the only moiety you can use on the N-terminus? Is a thioester the only activating group you can use on the C- terminus? Does the reaction go through a thioester intermediate?

29. Summary Peptide Synthesis Native Chemical Ligation Optimizing nucleophilic acyl substitution reactions Methodology Progress Future Work

30. Acknowledgments Paul Wentworth John Offer Mark Wormald Kavita Baruah Jason Chang Natalie Cygan Alicia Izharuddin Adria Lombardo Kanlaya Prapainop Johanna Scheinost Sarah Tully Daniel Witter

31. Questions or Comments