1. CONFORMATIONAL SPECIFIC SPECTROSCOPY OF JET COOLED 3-(4-HYDROXYPHENYL)-N-BENZYL- PROPIONAMIDE (HNBPA) ESTEBAN E. BAQUERO, V. ALVIN SHUBERT, AND TIMOTHY ZWIER Department of Chemistry, Purdue University West Lafayette, IN 47907

2. Introduction Motivation - Learn about Electronic Energy Transfer (EET) - Test Theories of Fluorescence Resonant Energy Transfer (FRET) measurements - Conformational specific spectroscopy allows us to test chromophoric distance and orientation dependence of EET. - Dispersed fluorescence measurements can be used to measure conformational efficiency of EET. HNBPA Flexible Rigid DeMember JR, Filipescu N. J. Am.Chem. Soc. 90, 6425 (1968)

3. Experimental methods Resonant 2 photon ionization (R2PI): Records spectra in mass selective fashion Biomolecule * (S 1 ) Biomolecule (S 0 ) Biomolecule + + e - Hole-burn Probe Conformer A Conformer B Hole-burn Probe R2PI: Electronic Spectrum UV-UV Hole-burning: Conformation specific electronic spectrum

4. P-Cresol 35338 cm -1 R2PI Spectrum of HNBPA

5. UV-UV Hole-burning Spectra of HNBPA -Two major conformers and one minor were found P-Cresol 35338 cm -1 Toluene 37477.4 cm -1 B A HB at 35670 cm -1 (also origin) HB at 35517 cm -1 (origin at 35368) Minor Conformer

6. Laser Induced Fluorescence Spectrum of p-cresol I Appel, K. Kleinermanns Ber Busenges. Phys. Chem. 91, 140-152 (1987)

7. UV-UV Hole-burning Spectra of HNBPA 6a 1 0 12 1 0 6a 2 0 12 2 0 6a 1 0 12 1 0 6a 2 0 S 2 0 0 0 Toluene-like origin Photon Energy (cm -1 ) A B

8. UV-UV Hole-burning Origin Region A B

9. R2PI Showing Progressions of Conformer A

10. 10.54 cm -1 20.10 cm -1 31.16 cm -1 38.80 cm -1 48.74 cm -1 66.29 cm -1 113.36 cm -1 9.69 cm -1 21.38 cm -1 31.35 cm -1 37.43 cm -1 49.56 cm -1 65.74 cm -1 100.25 cm -1 10.60 cm -1 18.07 cm -1 23.36 cm -1 31.29 cm -1 38.02 cm -1 57.62 cm -1 70.80 cm -1 9.62 cm -1 16.32 cm -1 22.67 cm -1 31.55 cm -1 40.43 cm -1 60.05 cm -1 74.77 cm -1 Fundamentals Conformer A 28cm -1 45 cm -1 111cm -1 Conformer B 16cm -1 34cm -1 38cm -1

11. 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 50-200 nsec IR Hole-burn UV probe Active Baseline Subtraction Wavenumbers (cm -1 ) Subtracted Signal UV only UV +IR Difference

12. IR Spectra of HNBPA N-H Stretch RegionO-H Stretch Region

13. Calculated Structures for HNBPA -Conformational search is done using AMBER force field in MacroModel package. - DFT calculations are then made using Gaussian03 at the B3LYP/6-31+G* level. 0.393 kcal/mol 0.313 kcal/mol 0.000 kcal/mol 0.143 kcal/mol0.884 kcal/mol 0.882 kcal/mol0.315 kcal/mol  0.400 kcal/mol Folded Perpendicular (FP) Extended Facing (EF) Extended Perpendicular (EP) Folded Facing (FF)

14. Calculated IR Hydride Stretch Region 0.000 kcal/mol 0.313 kcal/mol 0.882 kcal/mol 0.315 kcal/mol

15. UV-UV Hole-burning Spectra of HNBPA 6a 1 0 12 1 0 6a 2 0 12 2 0 6a 1 0 12 1 0 6a 2 0 S 2 0 0 0 Toluene-like origin Photon Energy (cm -1 ) A B

16. Summary Evidence Conformer A Conformer B UV Spectrum FP EP>EF≈FF IR Spectrum FPFF>EP S2 000S2 000S2 000S2 000FFEP>EF≈FF Tentative Assignment FPEP Folded Perpendicular (FP) Extended Facing (EF) Extended Perpendicular (EP) Folded Facing (FF)

17. Future Work -Conformational assignments by double resonant IR-microwave spectroscopy (collaboration with Brian Dian and Brooks Pate at the University of Virginia). TA06, TA07, TA08, TI09, TI11, RI06, FC03, FC04. - Dispersed fluorescence spectra will be taken in order to probe (EET) and its conformational dependence. - New systems will be studied such as: N-Z-L Serine benzyl ester

18. Conclusions - Electronic energy transfer could be tested in bichromophore systems such as HNBPA were the system’s flexibility allows the sampling of many distances and orientations between chromophores. - HNBPA may already show evidence for conformational dependence (EET), which needs further study. HNBPA Flexible

19. Acknowledgements People Prof. Timothy S. Zwier Prof. Timothy S. Zwier The Zwier Group Alvin Shubert Alvin Shubert Jasper Clarkson Jasper Clarkson Tracy LeGreve William “Bill” James Jaime Stearns Nathan Pillsbury Josh Newby Talitha Selby Funding National Science Foundation