1. Maria Eugenia Sanz, Carlos Cabezas, Santiago Mata, José L. Alonso The Rotational Spectrum of Tryptophan

2. Motivation 1986 6 conformers, REMPI No rotational spectrum 2000 3 conformers, REMPI & hole-burning 2001 6 conformers, IR ion-dip & hole-burning 2003 3 conformers, IR action spectroscopy 2009 7 conformers, cavity ring-down Previous spectroscopic studies of tryptophan 5 conformers assigned to specific structures Rizzo et al. J. Chem. Phys. 84, 2534 (1986) Piuzzi et al. Chem. Phys. Lett. 320, 282 (2000) Snoek et al. PCCP 3, 1819 (2001) Bakker et al. Phys. Rev. Lett. 203003-1 (2003) Rouille et al. J.Phys.Chem. A 113, 8187 (2009)

3. Motivation Laser ablation + MB-FTMW successful to study aliphatic amino acids Can it be applied to aromatic amino acids? Laser Solid sample Phenylalanine, tyrosine Tryptophan Very weak spectra o 60-80 mJ/pulse o Nd:YAG laser @ 532 nm o 5.5 bar Ne

4. Preliminary results Presented at Ohio in 2009 8 0,8  7 0,7 I’, F’ ← I’’, F’’ 2,10 ← 2,9 1,9 ← 1,8 One conformer observed ! 500 cycles

5. Experimental problems Photofragmentation Modification of laser ablation parameters 5000 ps 150 ps35 ps @ 355 nm 532 nm o Laser frequency o Laser pulse length 355 nm @ 5 ns S/N = 3 S/N = 4 S/N = 6 S/N = 20 250 cycles 500 cycles

6. Experimental problems Photofragmentation Modification of laser ablation parameters Small rotational constants Use of new spectrometer optimised for 2-10 GHz o Mirrors of 70 cm diameter o Curvature radius 70 cm Fabry-Pérot resonator

7. Experimental problems Photofragmentation Modification of laser ablation parameters Small rotational constants Use of new spectrometer optimised for 2-10 GHz

8. Experimental problems Photofragmentation Quadrupole coupling of two 14 N Modification of laser ablation parameters Use of isotopically enriched samples Small rotational constants Use of new spectrometer optimised for 2-10 GHz 14 N a - 15 N i 15 N a - 15 N i N N

9. Methods Experimental Laser ablation: 355 nm, 35 ps, 1-5 mJ/pulse Carrier gas Ne @ 15 bar 0.3 s MW pulse, 500s mol. pulse 14 N a - 14 N i sample one rotamer observed, lines from another one? Timeline 14 N a - 15 N i sample two rotamers observed, possibly three? 15 N a - 15 N i sample two rotamers confirmed

10. Methods Computational Intramolecular hydrogen bonds: Orientation of side chain: C COOH C  C  C  = +60° (a), -60° (b), 180° (c) type I type II type III N ― H···O=C N···H ― O N ― H···O-H Orientation of indole ring: C  C  C  C= +90° (+), -90° (-) 1. B3LYP/6-311++G(d,p) 2. MP2/6-311++G(d,p) Structure optimizations and vibrational frequency calculations Structure optimization on B3LYP geometries CC CC CC Starting configurations Calculations

11. Methods Computational Ib+Ib-IIb+IIb-IIIb+IIIb-Ia+Ia- Ic+1Ic+2Ic+3Ic-2IIc+1IIc+2IIc-1IIc-2IIIc+ 3393310444668515757745 cm -1 12259491020119485151310957271649 cm -1 MP2/6-311++G(d,p)

12. Rotational Spectrum Rotamer I experimental MP2 IIb+

13. Rotational Spectrum Rotamer II experimental MP2 ( 15 N i - 14 N a )

14. Rotational Spectrum Rotamer II experimental MP2 ( 15 N i - 14 N a ) IIc+1

15. Conclusions Two conformers of tryptophan identified in rotational spectrum IIb+IIc+1 Type II (N−H···O) hydrogen bonds preferred, in contrast with aliphatic amino acids Conformational behaviour follows that of phenylalanine Lee et al., J. Phys. Chem. A, 108, 69 (2004) Pérez et al., J. Phys. Chem. A, 115, 9253 (2011)

16. Conclusions Two conformers of tryptophan identified in rotational spectrum IIb+IIc+1 Sanz et al., J. Chem. Phys. 140, 204308 (2014) Snoek et al., PCCP, 3, 1819 (2001)

17. Acknowledgements Thank you for your attention Prof. Dr. Jens-Uwe Grabow, Hannover University MB-FTMW control software Funding Grupo de Espectroscopia Molecular Isabel Peña Susana Blanco Juan Carlos LópezLucie Kolesnicová Celina BermúdezAgustín Martín Vanesa VaqueroCristóbal Pérez