Spectroscopic investigation of β-peptides: Ac-β 3 -Phe-NHMe, Ac-β 3 -Phe-β 3 -Ala-NHMe and Ac-β 3 -Ala-β 3 -Phe-NHMe. Soo Hyuk Choi and Samuel H. Gellman Department of Chemistry, University of Wisconsin-Madison, Madison, WI Esteban E. Baquero, William H. James, III,, Timothy S. Zwier, Department of Chemistry, Purdue University, West Lafayette, IN 47907

Peptide Containing Systems in the Gas Phase α-peptides have been extensively studied in the jet by several groups. Gas phase studies are advantageous in probing the conformational preferences of isolated molecules, and give the best connection to theory. Michel Mons β-peptides differ from α-peptides by an extra carbon linking the peptide groups. The extra linkage provides extra flexibility and a different set of conformational preferences. Conformational preferences of β-peptides are not as well known or understood. Angew. Chem. Int. Ed. 2007, 46, Angew. Chem. Int. Ed. 2006, 45, Mattanjah S. deVries M. Gerhards Molecular Physics, Vol. 103, No. 11–12, 10–20 June 2005, 1521–1529

Ac-β 3 -Phe-NHMe Ac-β 3 -Phe- β 3 -Ala-NHMe Ac-β 3 -Ala-β 3 -Phe-NHMe C ß H N 1 C    C 1 H 3 C O 1 C 2 N 2 CH 3 O 2 H 1 H 2 The conformational preferences and H-bonding properties of synthetic foldamers: Beta peptides Collaboration with Samuel Gellman and Soo-Hyuk Choi Univ. of Wisconsin-Madison C6 C8 C10 C12

Experimental Resonant Two-Photon Ionization spectroscopy (R2PI) Biomolecule * (S 1 ) Biomolecule (S 0 ) Biomolecule + + e - - Heat to raise vapor pressure of molecules - Molecules entrained in gas pulse (neon backing gas at 1-5 bar for these studies) - Collisional cooling to zero point vibrational levels - Heating ( °C) leads to concerns about thermal decomposition. The R2PI method gives mass analysis to confirm the spectrum is due to the molecule of interest.

UV-UV Hole-burning Spectroscopy - Records the UV spectrum of a single conformation free from interference from others present in the expansion Biomolecule * (S 1 ) Biomolecule (S 0 ) Biomolecule + + e - Hole-burn Probe Conformer A Hole-burn Probe Conformer B UV Hole-burn laser fixed: Provides  selectivity UV probe laser tuned Boltzmann distribution of conformers in the pre-expansion Collisional cooling to zero-point vibrational level B* C A C C B A A A C A A B C C A A B B B B B B B UV C Laser Timing nsec UV Hole-burn UV probe

Resonant 2 Photon Ionization (R2PI) and Hole Burning Spectrum of Betapeptide Ac-   -Phe-NHMe Cluster Band Masses Observed: Dimer and M+149 g/mol Cluster Band Masses Observed: Water 1 and Water 2 One dominant conformer Minor Conformer

S o Resonant Ion-dip Infrared Spectroscopy (RIDIRS) Biomolecule * (S 1 ) Biomolecule + + e - Biomolecule (A) NH or OH stretch (S 0, v=1) UV Source fixed: Provides  selectivity IR Source tuned Laser Timing nsec IR Hole-burn UV probe Active Baseline Subtraction Wavenumbers (cm -1 ) Subtracted Signal UV only UV +IR Difference

C6 C8 RIDIR Spectrum of Conformer A and B Ac-β 3 -Phe-NHMe 1.mol -1.cm -1 C ß H2H2 N 1 C   C 1 H 3 C O 1 C 2 N 2 CH 3 O 2 H 1 H 2 A. Aubry, M.T. Cung and M. Marraud J. Am. Chem. Soc. 1985, 107, A B N 1 H 1 …O 2 =C cm -1 N 2 H cm -1 N 2 H 2… O 1 =C cm -1 N 1 H cm -1 FT-IR Spectrum of beta-peptide chain L-Pro-L-Ala N 1 H cm -1 N 2 H cm -1 C6 C8?

C6(c) C6(b) C6(a) C8(a) C8(b) C8(c) Chromophore Position φ1φ1 θ1θ1 ψ1ψ1 H NN H OO Betatripeptide H-bonding architectures 3 types of C6 rings 5 types of C8 rings 3 chromophore positions

DFT (OPT)MP2 (SP)RIMP2 (OPT) C6(c)(1) C6(b)(1) C6(a)(1) C8(a)(3) C8(b)(3) C8(c)(3) Calculated Relative Conformer Energies Ac-  3 -Phe-NHMe Representative C6 structures Representative C8 structures C6(a)(1) most stable Many C8 structures in next tier

Representative C8 H-bonded NH Unusual C8’s with weak H-bond Dominant conformer = C6 Minor conformer = unusual C8 (return to after consider tripeptides) C6 (a)(1) C6 (b)(1) C8(b)(3) C8(d)(3) C8(a)(2) Comparing Expt. and Calculated IR spectra Ac-  3 -Phe-NHMe Acute angle Long distance

A B C D E * * * * * A B C D E C6 C8 C10 C12 RIDIR Spectrum of Conformer A - E of Ac-β 3 -Phe-β 3 -Ala-NHMe R2PI and HB Spectra of Betapeptide Ac-β 3 -Phe-β 3 -Ala-NHMe 5 conformations, 2 major, 3 minor Several unique H-bonding architectures UVHB Spectra

C6/C6 C6(a) C6(b) C8(a) C8(b) C8/C8 C10C12 C8/C12 2 → 1. 3 → 1 Double ring / double acceptor C6/C8 1 → 2, 3 → 2 C6 C8 1→22→31→22→3 3→22→13→22→1 1→31→33→13→1 Single ring structures Chromophore Position Double ring structures Betatripeptide H-bonding architectures

C6a/C6a (1) C6a/C8a (1) C8a/C8a (2) C12 (1) C10 (1) C8/C12 (2) DFT B3LYP/6-31+G* relative energies Ac-β 3 -Phe-β 3 -Ala-NHMe C6a/C6a Double ring lowest C6/C6 C6/C8 C8/C8 Double rings Double ring/ Double acceptor Single ring Double ring

C6a/C6a (1) C6b/C6a (1) C8a/C8b (3) C8a/C8a (3) Conformer B Conformer D Conformer C Comparing IR spectra with calculations Ac-β 3 -Phe-β 3 -Ala-NHMe Conformers B,D = C6/C6 double rings (B=one of major conformers) Conformer C = C8/C8 double ring Assignments:

C8/C12 (2) C10(a)(1) C10(b)(3) C6a/C8a (1) Conformer A Conformer E Comparing IR spectra with calculations Ac-β 3 -Phe-β 3 -Ala-NHMe C8 H-bonded NH now lower in frequency than C6

C6 C8 C10 C12 A R2PI and HB Spectra of Betapeptide Ac-β 3 -Ala-β 3 -Phe-NHMe RIDIR Spectrum of Conformer A - E of Ac-β 3 -Ala-β 3 -Phe-NHMe B C A B C D E 5 conformations: 2 major (A,B), 3 minor (C-E)

Phe-Ala C6/C6 double ring Phe-Ala C8/C8 double ring Phe-Ala C10 Phe-Ala C6/C8 C8/C12 A B D E Comparing single-conformation IR spectra Ac-β 3 -Ala-β 3 -Phe-NHMe A=C6/C6 double ring B=C10 single ring 2 major conformers D=C8/C8 double ring E= Double ring/double acceptor C= TBD (C8/C8 double ring???) 3 minor conformers Same as Ac-β 3 -Phe-β 3 -Ala-NHMeSimilar to Ac-β 3 -Ala-β 3 -Phe-NHMe

Conclusions Ac-β 3 -Phe-NHMe 2 conformations 1 major (C6) and 1 minor (Unusual C8) Unusual C8 creates a weaker than normal H-bonded structure This has to be due to a long H--O distance or an acute angle between peptides. C ß H N 1 C    C 1 H 3 C O 1 C 2 N 2 CH 3 O 2 H 1 H 2 5 conformations, 2 major, 3 minor 2 major conformations are C6/C6 and C10 3 minor conformations are C8/C8, C6/C6 and the possibility of a double acceptor conformer C6/C8 or C8/C12. Ac-β 3 -Phe- β 3 -Ala-NHMe Ac-β 3 -Ala-β 3 -Phe-NHMe 5 conformations, 2 major, 3 minor Assignments can be done by comparison to Phe-Ala arrengement. 2 major conformations C10 and C6/C6 3 minor C8/C8, C8/C12 or C6/C8 and one unusual conformer not yet assignned.

Acknowledgements Professor Timothy S. Zwier Current Group Members: Dr. Ching-Ping Liu Dr. Christian Müller William H. James III * V. Alvin Shubert Tracy LeGreve Nathan Pillsbury Joshua Newby Chirantha Rodrigo Joshua Sebree Former Group Members: Dr. Jaime Stearns Dr. Talitha Selby Dr. Jasper Clarkson Professor Samuel H. Gellman Soo-Hyuk Choi Funding Professor Kenneth Jordan Dr. Daniel Schofield