1. Conformation-Specific and Mass-Resolved, Infrared-Population Transfer Spectroscopy of the Model γ 2 -peptide Ac- γ 2 -hPhe-NHMe Evidence for the Presence of Intramolecular Amide-Stacking Contribution: WG13 William H. James III, Evan G. Buchanan, Christian W. Müller, Michael G. D. Nix, and Timothy S. Zwier Purdue University, West Lafayette IN 47907 Li Guo, and Samuel H. Gellman University of Wisconsin-Madison, Madison WI 53706 Department of Chemistry International Symposium on Molecular Spectroscopy 64 th Meeting June 22-26, 2009

2. The Big Questions Would an amide-stacked interaction would be attractive or repulsive? How strong would it be? Are there any circumstances in which amide-stacking could compete with amide-amide H-bonds that are so pervasive in proteins? Remainder of Protein/Peptide Intramolecular Amide-Amide NH --- C=O H-bond Remainder of Protein/Peptide Intramolecular Amide-Amide Stacking

3. Motivation Chemists are using foldamers to dictate folding propensities leading to unique secondary structures with biological relevance and efficacy. Goals Probe nearest-neighbor and next nearest-neighbor conformational preferences. Obtain single-conformation spectral signatures. Test current computational methods. Synthetic Foldamers T. S. Zwier, J. Phys. Chem. A 110 4133 (2006) S. H. Gellman, Acc. Chem. Res. 31 173 (1998) γ β α Ac- γ 2 -hPhe-NHMe Conformational Preferences = ? Ac-β 3 -hPhe-NHMe C6 (dominant) C8 (minor) Baquero et al. JACS 130 4784 (2008) Ac-α-Phe-NHMe Gerhards et al. PCCP 8 1660 (2004) C5 (dominant) C7 eq (minor) α β α James et al. submitted

4. Experimental Methods S1S1 S0S0 Biomolecule + + e - Ionization Continuum B TUNED UV PROBE Ionization Continuum A FIXED IR TUNED UV PROBE Ionization Continuum TUNED IR FIXED UV PROBE B A B A RESONANT TWO-PHOTON IONIZATION SPECTROSCOPY IR/UV HOLEBURNING SPECTROSCOPY RESONANT ION-DIP INFRARED SPECTROSCOPY Electronic Spectrum of ALL Conformers Conformation-Specific Electronic Spectrum Conformation-Specific IR Spectrum

5. Conformation-specific UV and IR Spectroscopy Amide I Spectral Region Amide NH Stretch Spectral Region A A B B C C Resonant Two-Photon Ionization Spectrum and IR/UV Holeburning Spectra A B C IR/UV holeburning shows the presence of three conformers. Two dominant conformers (A and B) have near identical signatures. Minor conformer (C) shows no H- bonding! A B γ β α

6. 1 2 3 4 5 6 7 8 9 A [C9(a)]B [C9(g-)] C [S(a)] Amide Stacked γ-peptide backbone Conformers of Ac- γ 2 -hPhe-NHMe DFT M05-2X/6-31+G(d) A B C A B C A B C C9(a) 0.97 kJ/mol C9(g-) 0.00 kJ/mol S(a) 1.31 kJ/mol α β γ Conformational Assignments Three conformers (A-C): Two dominant, one minor A and B: C9 hydrogen bond (differ by Phe position) C: Intramolecular Face-to-Face Amide-Stacked Motif No C7 hydrogen bonded conformers DFT M05-2X/6-31+G(d) C=O scaled 0.96, NH scaled 0.94

7. A Closer Look at Intramolecular Amide-Stacking Amide planes are nearly parallel Amide dipoles are anti-aligned C [S(a)] Amide-Stacked γ-peptide backbone DFT M05-2X/6-31+G(d) + - + - Amide-stacked structure is as low in energy as H-bonded conformers. Relative Energies of the Conformers of Ac- γ 2 -hPhe-NHMe Using Dispersion Corrected Density Functional Theory

8. The Nature of the Amide-Stacking Interaction DFT M05-2X/6-31+G(d) Systematic Fragmentation Method + Effective Fragment Potential Amide-stacked ~2/3 as strong as Amide-Amide H-bond! (Non-bonded energy) Increase in strain in trimethylene bridge of amide-amide H-bond conformers, while amide-stacked structure is well set-up to reduce strain! (Through Bond Energy) C [S(a)] Amide-Stacked γ-peptide backbone Large electrostatic and dispersion contributions. Chakraborty and Lim, J Phys Chem 99 17505 (1995) 1,3-diphenylpropane

9. A 55% C9(a) B 40% C9(g-) C 5% Amide- Stacked Requires assumptions taking into account R2PI cross sections, lifetimes and Franck-Condon Factors for each conformer. Does Amide-Stacking Effectively Compete with Hydrogen Bonding for Population? Fractional Abundances Using S 0 -S 1 Origin Intensities

10. Direct Experimental Measure of Fractional Abundances Mass-resolved, Infrared-Population Transfer Spectroscopy A B C Weighted Sum Amide NH Stretch Fluorescence based IR-PT Dian et al., J. Chem. Phys. 120 133 (2004) Dian et al., J. Chem. Phys. 120 9033 (2004)

11. A MRIRPT 38 + 1 % R2PI 55% C9(a) B MRIRPT 41 + 2 % R2PI 40% C9(g-) C MRIRPT 21 + 1 % R2PI 5% Amide- Stacked Amide-stacked population within a factor of 2 of amide-amide H-bonded conformers. Does Amide-Stacking Effectively Compete with Hydrogen Bonding for Population? Fractional Abundances via MRIRPT 1 2 3 4 5 6 7 8 9 A [C9(a)] 0.97 kJ/mol B [C9(g-)] 0.00 kJ/mol C [S(a)] 1.31 kJ/mol Amide-stacked γ -peptide backbone Conformers of Ac- γ 2 -hPhe-NHMe YES! The Amide-stacked motif effectively competes with H-bonded structures

12. Answers to the Big Questions Would an amide-stacked interaction would be attractive or repulsive? Yes! The amide-stacking interaction is an attractive interaction led by dispersion and electrostatic contributions. How strong would it be? The amide-stacking interaction is ~2/3 as strong as an amide-amide hydrogen bond (Non-bonded energy)! Are there any circumstances in which amide-stacking could compete with amide-amide H-bonds that are so pervasive in proteins? Yes! Amide-stacking not only competes with H-bonding (within a factor of 2 of H-bonded conformers) but is favored over some H-bonding motifs!

13. Amide-stacked + Bridging Water The Big Picture Amide-Stacking in Other Molecules doubly methylated derivative Water n=1 cluster Baldauf, et al. Angew. Chem. Int. Ed. 43 1594 (2004) Amide-stacking is present in small γ -peptides. Amide-stacked signature is spectrally overlapped with free amide NH stretches, therefore overlooked in solution studies. Anticipated that both H-bonding and Amide-stacking will occur in larger γ -peptides. Methyl substitution blocks C9 formation

14. Acknowledgements Professor Timothy S. Zwier Zwier Group Members Joshua Newby Chirantha Rodrigo Joshua Sebree James Redwine Zachary Davis Ryan Muir Deepali Mehta Dr. Jaime Stearns Dr. Jasper Clarkson Dr. Talitha Selby Dr. Esteban Baquero Dr. V. Alvin Shubert Dr. Nathan Pillsbury Dr. Tracy LeGreve FUNDING Professor Samuel H. Gellman Li Guo Department of Chemistry Professor Mark S. Gordon Luke Roskop THANK YOU ! γ-peptide synthesis SFM/EFP Calculations