Maria Eugenia Sanz, Carlos Cabezas, Santiago Mata, José L. Alonso

The Rotational Spectrum of

Tryptophan

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)

Motivation

Laser ablation + MB-FTMW successful to study aliphatic amino acids

Can it be applied to aromatic amino acids?

Laser

Solid sample

Phenylalanine, tyrosine

TryptophanVery weak spectra

o 60-80 mJ/pulse

o Nd:YAG laser @ 532 nm

o 5.5 bar Ne

5783.2 5783.4 5783.6 5783.8 5784.0

0.0

0.5

1.0

Rel. int.

(MHz)

Preliminary results

Presented at Ohio in 2009

80,8 70,7

I’, F’ ← I’’, F’’2,10 ← 2,91,9 ← 1,8

HN

O

OH

NH2

One conformer observed !

500 cycles

Experimental problems

Photofragmentation Modification of laser ablation parameters

5000 ps 150 ps 35 ps @ 355 nm

532 nm o Laser frequency

o Laser pulse length

355 nm @ 5 ns

S/N = 3 S/N = 4

S/N = 4 S/N = 6 S/N = 20

250 cycles500 cycles500 cycles

500 cycles 500 cycles

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

Experimental problems

Photofragmentation Modification of laser ablation parameters

Small rotational constants Use of new spectrometer optimised for 2-10 GHz

5783.2 5783.4 5783.6 5783.8 5784.0

0.0

0.5

1.0

Rel. int.

(MHz)

5783.2 5783.4 5783.6 5783.8 5784.0

0.0

0.5

1.0

Rel

. in

t.

(MHz)

Experimental problems

Photofragmentation

Quadrupole coupling of two 14N

Modification of laser ablation parameters

Use of isotopically enriched samples

Small rotational constants Use of new spectrometer optimised for 2-10 GHz

14Na-15Ni

15Na-15Ni

HN

O

OH

NH2NN

Methods

Experimental Laser ablation: 355 nm, 35 ps, 1-5 mJ/pulse

Carrier gas Ne @ 15 bar

0.3 ms MW pulse, 500ms mol. pulse

• 14Na-14Ni sample one rotamer observed, lines from another one?

Timeline

• 14Na-15Ni sample two rotamers observed, possibly three?

• 15Na-15Ni sample two rotamers confirmed

Methods

Computational

• Intramolecular hydrogen bonds:

• Orientation of side chain: CCOOHCaCbCg = +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: CaCbCgC= +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

HN

O

OH

NH2

Ca

CbCg

Starting configurations

Calculations

Methods

Computational

Ib+ Ib- IIb+ IIb- IIIb+ IIIb- Ia+ Ia-

Ic+1 Ic+2 Ic+3 Ic-2 IIc+1 IIc+2 IIc-1 IIc-2 IIIc+

339 331 0 444 668 515 757 745 cm-1

1225 949 1020 1194 851 513 1095 727 1649 cm-1

MP2/6-311++G(d,p)

Rotational Spectrum

Rotamer Iexperimental MP2

IIb+

14Ni-14Na

15Ni-14Na

15Ni-15Na I I b+ I a- I b+

A (MHz) 1243.5844(29) 1231.0742 (24) 1219.484(7) 1244 1144 1171 B (MHz) 392.48409(11) 392.15526(17) 391.33142(8) 396 403 419 C (MHz) 346.88467(16) 345.65584(22) 344.31779(9) 349 362 362

N

amino aa (MHz)

[0.31]

[0.31]

-

0.31

2.31

1.35

bb (MHz) 1.714 1.777 - 1.97 -1.01 -0.55 cc (MHz) -2.024 -2.087 - -2.28 -1.30 -0.80 bb - cc (MHz) 3.739(41) 3.865(17) -

N

indole aa (MHz)

1.079(74)

-

-

1.01

1.02

0.96

bb (MHz) 1.301(55) - - 1.37 1.78 1.57 cc (MHz) -2.380(55) - - -2.39 -2.80 -2.53

EMP2 (cm-1) 0 745 339 G (cm-1) 0 245 468

Rotational Spectrum

Rotamer IIexperimental MP2 (15Ni-14Na)

15Ni-14Na

15Ni-15Na I c+2 I c+3 I I c+1 I I c+2

A (MHz) 1281.310(15) 1272.455(8) 1247 1253 1238 1273 B (MHz) 333.70096(14) 332.40803(8) 342 336 330 338 C (MHz) 287.10022(17) 286.33178(9) 299 292 291 290

N

amino aa (MHz)

-2.33(18)

-

1.21

1.27.31

-2.52

-2.42

bb (MHz) 1.95(12) - 1.13 1.75 1.99 0.20 cc (MHz) 0.38(12) - -2.35 -3.02 0.53 2.23 bb - cc (MHz) 1.573(62) -

EMP2 (cm-1) 0 1020 851 513 G (cm-1) 414 448 70 84

Rotational Spectrum

Rotamer IIexperimental MP2 (15Ni-14Na)

15Ni-14Na

15Ni-15Na I c+2 I c+3 I I c+1 I I c+2

A (MHz) 1281.310(15) 1272.455(8) 1247 1253 1238 1273 B (MHz) 333.70096(14) 332.40803(8) 342 336 330 338 C (MHz) 287.10022(17) 286.33178(9) 299 292 291 290

N

amino aa (MHz)

-2.33(18)

-

1.21

1.27.31

-2.52

-2.42

bb (MHz) 1.95(12) - 1.13 1.75 1.99 0.20 cc (MHz) 0.38(12) - -2.35 -3.02 0.53 2.23 bb - cc (MHz) 1.573(62) -

EMP2 (cm-1) 0 1020 851 513 G (cm-1) 414 448 70 84

IIc+1

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 phenylalanineLee et al., J. Phys. Chem. A, 108, 69 (2004)Pérez et al., J. Phys. Chem. A, 115, 9253 (2011)

Acknowledgements

Thank you for your attention

• Prof. Dr. Jens-Uwe Grabow, Hannover UniversityMB-FTMW control software

Funding

• Grupo de Espectroscopia Molecular

Isabel Peña Susana Blanco

Juan Carlos LópezLucie Kolesnicová

Celina Bermúdez Agustín Martín

Vanesa Vaquero Cristóbal Pérez