1. Probing the Electronic Structure of Peptide Bonds using Methyl Groups: Experimental Measures of Resonance Weights a Physics Lab, NIST, Gaithersburg, MD b Chemistry Dept., University of Pittsburgh, PA David F. Plusquellic a and David W. Pratt b

2. Principal Resonance Structures of Peptide Bond E = 30 kcal/mole * sin 2 ω 60%40% 1.33 Å L. Pauling et al., Proc. Natl Acad. Sci. USA 37, 205-211 (1951). R. B. Corey and L. Pauling, Proc Roy. Soc B 141, 10-20 (1953) 1.24 Å

3. Outcome I: Assigned Conformational Structures to > 10 systems Rotor axis angles, inertial constants and dipole moments Outcome II: Determined V 3 Barriers V 3 barriers vary by more than 2-fold and therefore, sensitive to changes in structure that occur at the other end of the peptide bond. Issues addressed What are the principal factors responsible for the torsional barriers? What influence do torsional motions have on relative weights of resonance structures I and II? Carbonyl Methyl Amide Methyl Gas Phase FTMW Studies of Methyl Terminated Model Peptides

4. CSCS C1C1 N-MethylAcetamide MA t N-AcetylGlycine AG I N-AcetylGlycineEthylEster EAA I & EAA II Proline Dipeptide PD tCd Model Peptides Investigated Alanine Dipeptide AD 7eq [J. Mol. Spec., 227, 28 (2004)] [J. Mol. Spec., 228, 251 (2004)] [J. Chem. Phys., 119, 5497 (2003)] [J. Chem. Phys., 118, 1253 (2003)] [in preparation]

5. Summary of the Experimentally Determined V 3 Barriers Torsional Barriers have their origin in electronic structure of peptide bond

6. Methyl Acetamide - MA t anti syn anti syn MP2/aug-cc-pVTZ Predictions Syn forms found to be more stable in previous theoretical work on biacetyl, propene, acetaldehyde, etc.* * D. W. Pratt, et al., J. Am. Chem Soc. 109, 6591 (1987) J. A. Pople, et al., J. Am. Chem Soc. 98, 664 (1976) What level of theory is needed? CHARMm, MM AM1 Force Field and Semiempirical Models CHARMm, MM

7. Level of Theory HF/6-311++G(d,p) chosen level V3EV3E anti V 3 = E syn - E anti V3TV3T anti syn

8. Natural Bond Orbitals or NBOs Prof. Frank Weinhold, http://www.chem.wisc.edu/~nbo5 1.Lewis interactions arising from electrostatic and exchange repulsion interactions of the NBOs of the idealized Lewis structure Molecular e - density is decomposed in terms of bonding and anti-bonding orbital densities. Convenient way to separate energies associated with F is the Fock operator and ε σ and ε σ* are NBO orbital energies Donor-Acceptor Interaction Energies from Second-Order Perturbation Theory Rigorous separation made by deleting all anti-bonding and Rydberg orbitals and calculating new SCF energy 2. Non-Lewis stabilizing interactions arising from delocalization of the NBOs into empty Rydberg and antibonding (non-Lewis) orbitals

9. Lewis vs non-Lewis Contributions - HF/6-311++G(d,p) E non-Lewis E Lewis E Opt 0  E non-Lewis- =  E Opt -  E Lewis E Lewis E Opt synanti V3V3 { Amide E Lewis E Opt 0  E non-Lewis- =  E Opt -  E Lewis E Lewis E Opt E non-Lewis antisyn V3V3 } Carbonyl Lewis Energy (LE) differences always favor syn configuration Non-Lewis (NLE) differences always favor anti configuration

10. What are the principal contributions to the Lewis and non-Lewis energies differences? Why are the Lewis and non-Lewis interactions reversed? Torsional Dependence of Lewis vs non-Lewis Differences

11. σ C-H 3σ C-H σ C-N π C=O σ C=O σ N-H Lewis Interactions - Bonding PNBOs

12. Vicinal Lewis Orbital Energies – Carbonyl Methyl Vicinal Bonds σ C-H ip - σ C=O anti syn

13. Remote Lewis Orbital Energies - Carbonyl Methyl Remote Bond Remote Orbitals n O (s-like) n O (p-like)

14. Lewis Orbital Energies – Amide Methyl Remote Bonds Remote Orbitals Vicinal Bonds Vicinal Orbital

15. Lewis Energy Difference Summary Explains - the syn preference for amide methyl: vicinal Lewis energy differences dominate - the higher sensitivity of carbonyl methyl barriers to remote structural changes

16. Non-Lewis Interactions – PNBO Delocalizations σ N-H * σ C-H π C=O * 3σ C-H σ C=O * σ C-N *

17. Non-Lewis Energy Differences – Carbonyl Methyl AG I anti σ C-H  σ C=O * syn σ C-H  σ C=O *

18. Non-Lewis Energy Differences – Amide Methyl AD 7eq NLE differences do not arise from direct interactions with the methyl top orbitals

19. Principal Delocalization: n N  π C=0 * Indirect consequence of the methyl group interactions Order of magnitude larger than any direct NLE σ C-H  σ C=O * 5 kcal/mole n N  π C=0 * 100 kcal/mole

20. Non-Lewis Energy Difference Summary NLE difference should be related to the magnitude of the charge shifts

21. Torsional Dependence of Natural Charges Natural charge shifts reflect changes in resonance character Decrease in n N  π C=0 * Increase in n N  π C=0 *

22. Natural Resonance Theory Resonance weights decomposed from electron density 1-e density operator represented in terms of an optimized resonance hybrid of density operators Each optimized resonance density is the determinant of the doubly occupied NBO for the chosen Lewis structure. I II III – + – +

23. Resonance Structural Weights Carbonyl Amide

24. Resonance Structural Weights III C II N dipolarcovalent

25. Resonance Structural Weights III C II N dipolar covalent

27. “Infinite-Barrier” Rotational Constants A-state E-state Experimental Evidence of Resonance Weight Changes

28. Separate Fit Cases using the “high-barrier” Approximation No improvement upon including - 4 th order terms - denominator corrections

29. At top-of-barrier, A-state has ~7% larger probability amplitude than E-state Semi-Quantitative Agreement

30. Internal rotation models will need to include additional degrees of freedom to account for resonance character changes

31. Conclusions of MW Studies Nine methyl torsional barriers have been investigated for 5 different model peptides Non-Lewis energy differences favor the anti configuration Lewis energy differences favor the syn configuration Both are important resulting in low V 3 barriers Carbonyl Methyl Groups Non-Lewis energy differences dominate – anti minimum. Local steric interactions tend to cancel Amide Methyl Groups Lewis energy differences dominate - syn minimum Local steric interactions are most important

32. Acknowledgements Richard Lavrich, EPA Michael Tubergen, Prof., Kent State Univ. Jon Hougen, Richard Suenram, Frank Lovas, Gerald Fraser and Angela Hight-Walker, NIST Isabelle Kleiner, CNRS, Paris, France Frank Weinhold, Prof., Univ. of Wisconsin

34. Semi-Quantitative Agreement

36. Carbonyl E-state V 3 T has more covalent character (I) than V 3 E Amide E-state V 3 T has more ionic character (II) than V 3 E

37. Non-Lewis energy differences increase at the MP2/6-311++G(d,p) Carbonyl Methyl anti syn II Amide Methyl anti syn V 3 increasesV 3 decreases I I II

44. I I

45. Gas Phase FTMW Studies of Methyl Terminated Model Peptides Outcome I: Assigned Conformational Structures to > 10 systems Rotor axis angles, inertial constants and dipole moments Outcome II: Determined V 3 Barriers V 3 barriers vary by more than 2 fold and therefore, sensitive to changes in structure that occur at the other end of the peptide bond. Carbonyl MethylAmide Methyl Issues addressed What are the principal factors responsible for the torsional barriers? What influence do torsional motions have on relative weights of resonance structures I and II?

46. O Primary Peptide Structure - Biochemistry Primer Primary structures Alanine - Amino acid Dialanine - Polypeptides + = + Water N C H Ramachandran Angles ψ φ

47. α-Helix Parallel β-Sheet Anti-parallel β-Sheet Secondary Peptide Structure - Biochemistry Primer φ ≈-140º ψ ≈+140º φ ≈57º ψ ≈47º