Simultaneous Ionization-Detected Stimulated Raman and Visible

Letter pubs.acs.org/JPCL

Simultaneous Ionization-Detected Stimulated Raman and Visible− Visible−Ultraviolet Hole-Burning Spectra of Two Tryptamine Conformers Nitzan Mayorkas, Shay Izbitski, Amir Bernat, and Ilana Bar* Department of Physics, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ABSTRACT: A key first step toward probing structures and interactions of individual conformers of isolated flexible molecules is uncovering their characteristic spectral signatures. Here, conformation-specific ionization-detected stimulated Raman (IDSR) and visible−visible−ultraviolet hole-burning spectra were measured simultaneously to determine the unique signatures of the two most stable conformers of tryptamine in the gas phase. These signatures together with the comparison of the IDSR spectra to the computationally predicted Raman spectra assisted in their characterization and structural identification. This new approach offers high selectivity and is foreseen to be an improved diagnostic tool for dissection of conformers of flexible molecules.

SECTION: Kinetics, Spectroscopy

C

processes13,14 or by the Free Electron Laser for Infrared eXperiments (FELIX),15−17 respectively. In ILSRS, stimulated Raman pumping is followed by ionization probing of the depleted population in the lower level of the Raman transition (vibrational ground state). The depletion is induced by two coherent incident laser fields (the pump, ωp, and Stokes, ωS), which drive the Raman scattering in the probed molecules. A great advantage of ILSRS is that, similarly to IR-IDS, it offers the possibility to investigate isolated molecules or clusters in molecular beam experiments by using two different visible (vis) laser beams to monitor the Raman spectrum in a broad spectral range, with uniformly high resolution, and based on tabletop laser equipment. In an attempt to expand the capability of ILSRS for conformational studies, we present in this Letter the first ILSR spectra providing direct evidence for the shapes of the two most stable tryptamine (TRA) conformers in the gas phase. TRA is a potent neurotransmitter or neuromodulator, containing a flexible ethyl amino side chain fused to an indole ring. It has been chosen as a model system because it has been intensively studied by a variety of pioneering experiments to resolve the issue of its conformational assignment.18−31 These studies resulted in a wealth of information on its seven isolated conformers, with which our data can be compared. Interestingly, in this study, the photons driving the SR process could sample portions of the electronically excited potential energy surface via competing routes, leading to additional

haracterization of three-dimensional structures of biologically relevant molecules, their dynamical flexibility to fold into unique structures, and their inter- and intramolecular interactions with each other and with the ubiquitous solvent water are initial key points to be addressed to reveal an understanding of the functioning and behavior in actual biological environments.1−7 Recently, significant advances in spectroscopic techniques have opened a window for the conformational analysis of isolated molecules in the gas phase. Particularly, double resonance methods based on vibrational spectroscopies, including infrared ion dip spectroscopy (IR-IDS),1−7 have been found to be powerful structural probes. Nevertheless, methods that have been used to monitor Raman spectra of different species in very narrow spectral ranges, including stimulated Raman−UV double resonance spectroscopy combined with fluorescence detection8 and ionization-loss stimulated Raman spectroscopy (ILSRS),9,10 could be foreseen as potential conformational probes. Indeed, very recently, we have developed ILSRS11,12 for the measurement of conformation-specific ILSR spectra in extended ranges. The IR-IDS and ILSRS methods take advantage of the inherent mass and wavelength selectivity of the resonantly enhanced two-photon ionization (R2PI) process, offering the possibility to monitor the ion signal from specific conformers. The vibrational spectra of each conformer can then be studied by monitoring the IR- or Raman-induced depletion in the ion signal as a function of the frequency of the laser affecting the vibrational excitation. Because IR radiation is usually generated by nonlinear processes, in most IR-ID spectra, only hydride stretches are measured. In some experiments, the amide mode region and the modes in the 500−1800 cm−1 frequency range were accessed by IR radiation generated by nonlinear © 2012 American Chemical Society

Received: January 7, 2012 Accepted: February 7, 2012 Published: February 7, 2012 603

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interfering lines in the spectra. The questions that immediately arose were, What is the origin of these lines? Furthermore, could these lines together with the ILSR signatures be leveraged to further assist the characterization and identification of the sampled conformers? The TRA (98% purity) was purchased from Sigma-Aldrich Co. and used without further purification. A stream of argon at ∼4 bar of pressure was passed through a pulsed nozzle valve and then through the TRA vapor, held in a heated reservoir (∼110 °C). The seeded gas passed through a tip before expansion into the vacuum chamber of a home-built time-offlight mass spectrometer (TOFMS). The molecular beam was intersected by the laser beams under the tip exit. Mass-selected R2PI spectra of TRA were recorded using a tunable frequency-doubled pulsed neodymium:yttrium aluminum−garnet (Nd:YAG)-pumped dye laser (UV) with ∼5 ns pulses at a 10 Hz frequency. The conformer-specific spectra of TRA were subsequently recorded by three-color vis−vis−UV excitation. These spectra were measured using two different vis beams, ωp and ωS, inducing the vibrational excitation via SRS while tuning the UV laser onto a selected absorption band. In these experiments, a second Nd:YAG laser provided the second harmonic (532 nm) with ∼5 ns pulses at 10 Hz. This green beam was split in a ratio of four to one, so that the more intense beam pumped a dye laser to generate the tunable ωS beam and the second beam provided ωp. The vertically polarized ωp and ωS beams, with energies of ∼13 mJ, were counterpropagated and temporally and spatially overlapped to drive the observed Raman transitions close to saturation. The SRS beams were aligned to spatially overlap the UV beam in the interaction region and to precede the UV beam by ∼30 ns. Ions formed via R2PI and three-color vis−vis−UV excitation were detected and fed into a fast preamplifier, allowing measurement of the MS with a fast digital oscilloscope and recording of the integrated intensity of the m/z = 160 ion peak using a recently developed application on the LabView software. The spectra were monitored by measuring the integrated ion signal as a function of the UV or ωS beam wavelengths. For each conformer, eight spectra were measured and averaged to achieve suitable signal-to-noise levels. Following the previously described approach,11,12 analysis of the spectra was performed by coupling the experimental results with those from quantum mechanical, density functional theory (DFT), and ab initio calculations, using the GAUSSIAN 09 package,32 providing the structures of the different conformers and the calculated Raman spectra. Optimization of the geometries of nine conformers of TRA by the Becke threeparameter hybrid functional combined with Lee−Yang−Parr correlation functional (B3LYP)33,34 and the 6-311++G(d,p) basis set was performed. Also, single-point energy calculations at the found minimum energies were carried out by secondorder Møller−Plesset theory (MP2) and the same basis set. The revealed geometries were used for calculation of harmonic and anharmonic vibrational frequencies and Raman activities at the B3LYP/6-311++G(d,p) level. The R2PI spectrum that was monitored revealed the same six features that were previously observed,21,26,27,30 with band A being the most intense and the highest in energy (∼34 915 cm−1). The other bands appeared at the low-frequency side of A with B being lower by ∼20 cm−1. The spectra obtained in the three-color vis−vis−UV experiment, when the UV laser was parked on the A and B bands, are shown in trace (i) of Figure 1A and B, respectively. It can be clearly seen that in addition to

Figure 1. (A, B) Respective spectra of the A and B conformers of tryptamine: (i) ionization-loss stimulated Raman, including simultaneously measured features by two-color vis−vis−UV hole burning as well as by ionization-gain stimulated Raman in (B); (ii) two-color vis− vis−UV hole burning; (iii) the “net” ionization-detected stimulated Raman; and (iv) below the net spectrum, a theoretical Raman spectrum that exhibits the closest agreement is displayed. The lines in these spectra are based on anharmonic vibrational frequencies and Raman intensities calculated at the B3LYP/6-311++G(d,p) level and convoluted with Lorentzian lines of full width at half-maximum (fwhm) of 0.5 cm−1. The dashed red lines are a guide to the eye, used to mark features in the ILSR spectrum of conformer A that are observed in the Raman gain spectrum. The electronic ground state, S0, structures of GPy(out) and GPy(up), A and B conformers of tryptamine, optimized at the B3LYP/6-311++G(d,p) level of theory are shown in the right parts of panels (A) and (B), respectively.

the constant ion background level, very sharp features pointing downward at particular Raman shifts appear in the spectra. These features result from the SR-induced depletions in the population of the lower level of the Raman transition as a function of the ωS wavenumber. Nevertheless, careful examination of these spectra shows that together with these sharp features, extra lines appear, some broad and pointing down, probably due to a different depletion process, as well as some very narrow lines pointing up [see Figure 1B(i)]. Puzzled by the issue of what could be the origin of these extra lines, we hypothesized that the broad ones are a result of double resonance two-color vis−vis−UV hole burning, while the sharp ones are due to the other variant of mass-selective ionization-detected stimulated Raman (IDSR),9,10,35 that is, ionization-gain stimulated Raman (IGSR). The option of twocolor hole burning was operationally tested by fixing the UV laser on the transition corresponding to the particular conformer, that is, on the A or B bands, while blocking the ωp beam and scanning the ωS beam across the respective wavelength range. In these experiments, the spectra shown in Figure 1A(ii) and B(ii) were obtained. These spectra correspond to vis−vis resonant transitions, depleting the population of vibrationless ground-state molecules and eventually reducing the UV signal from the ground state and consequently allowing sampling of the excited potential energy surface in the 34 250−36 750 cm−1 range. It is immediately apparent that a very good matching exists between the features in the hole-burning spectra of conformers A and B of TRA (see below), in Figure 1A(ii) and B(ii), and the broad lines observed in Figure 1A(i) and B(i). This implies that the broad lines in 604

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the three-color vis−vis−UV spectra are indeed a result of twocolor vis−vis−UV hole burning, where their frequencies, shapes, and intensities vary from one conformer to another. Table 1 lists the frequencies of the vibronic transitions in each Table 1. Transition Frequencies of Two of the Conformers of Tryptamine Obtained by Two-Color vis−vis−UV HoleBurning Spectroscopy origin

vibronic frequency (cm−1)

A

34 919 35 334 35 499 35 519 35 658 35 891 36 241 36 389 36 453 34 899 35 438 35 523 35 624 35 657 35 746 36 146 36 386 36 441

B

Figure 2. (A, B) Expanded portions of the respective spectra of conformers A and B of tryptamine: (i) ionization-loss stimulated Raman including simultaneously measured features by two-color vis− vis−UV hole burning as well as ionization-gain stimulated Raman in (B) and (ii) the corresponding calculated Raman spectra at the B3LYP/6-311++G(d,p) level.

the subtle differences in their structures. By comparing these highly informative IDSR spectra to the calculated Raman spectra of the seven conformers, we found that the spectra displayed in trace (iv) of Figure 1 and in trace (ii) of Figure 2 show the best overall agreement with the measured ones. For example, the multiple peak structure in the region of 750−800 cm−1 in the two conformers is nicely matched by the calculated spectra. This implies that these IDSR spectra correspond to the A and B conformers of TRA, representing structures differing by the amino group orientation (see structures in the right portion of Figure 1A and B). According to the previously introduced nomenclature,21 conformer A is compatible with GPy(out), and B is compatible with GPy(up), and our computations using the B3LYP and MP2 methods with the 6-311++G(d,p) basis indicate that the zero-point corrected electronic energies of conformer A are favored over those of B by 72.7 and 126 cm−1, respectively. These findings are consistent with previous assignments of the R2PI spectrum and measurements in the 3 μm IR range.21,26,27,29 The observation of the isolated A and B conformers of TRA is also supported by the uniquely detailed studies of Zwier and co-workers23,24 on the energy thresholds for conformational isomerization, which gave evidence for the existence of the TRA conformers and also characterized their dynamics. In these studies, stimulated emission pumping hole-filling spectroscopy (SEP-HFS) and stimulated emission pumping population transfer spectroscopy (SEP-PTS) were used to study the conformational isomerization of TRA. The results of these measurements together with those of quantum chemical calculations pointed to bounds for isomerization of conformer A to B of 688−748 cm−1 and to the fact that B is situated at least 126 cm−1 above A. As can be seen from the structures shown in Figure 1A and B, conformers A and B differ primarily in the orientation of the NH2 group, and therefore, it was suggested23,24 that tunneling might play a role in the isomerization pathway connecting them. Interestingly, Nguyen

of these conformation-specific vis−vis−UV hole-burning spectra. The band origins and the frequencies of some features correspond to the vibronic transitions measured by UV−UV hole burning,21 implying that this conformer-specific information, obtained simultaneously with the IDSR lines, could be used to support the conformer's identification. By subtracting the vis−vis−UV hole-burning spectra, trace (ii), from the corresponding spectra including the simultaneously measured IDSR and vis−vis−UV features, trace (i), the “net” IDSR spectra (iii) for conformers A and B are obtained. The IDSR spectrum of the less populated conformer (the UV laser parked on band B of the R2PI spectrum), shown in Figure 1B(iii), shows in addition to the ILSR lines, sharp Raman gain lines due to ionization probing of the upper Raman-pumped level. It is clearly seen that most IGSR lines in Figure 1B(iii), marked by the dashed red lines, correspond to the ILSR lines of the most populated conformer, Figure 1A(iii). This increase in the photoionized species, affected by resonant R2PI probing of excited SR transitions, leads to enhancement of the massselected ion signal as a function of the ωS frequency, allowing clear observation of some features in favorable cases. For example, the lines appearing at 697, 757, 871, 1010, 1016, 1332, and 1362 cm−1 are prominent in the IGSR spectrum of Figure 1B(iii), providing additional evidence for the existence of the ILSR features, Figure 1A(iii), and eventually can assist in the characterization of the conformers. Although, at first glance, it seems that many of the lines in the IDSR spectra, traces (i) and (iii) of Figure 1A and B, have identical Raman shifts, careful examination shows that this is not the case. This is even more apparent in the expanded portions of the three-color vis−vis−UV spectra (650−1250 cm−1), Figure 2A(i) and B(i), which show that the ILSR and IGSR features corresponding to conformer A are slightly shifted in positions relative to the ILSR lines of conformer B, reflecting 605

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and Pratt26,27 suggested that the splittings in the highresolution electronic spectra of some TRA conformers were also produced by tunneling motions. To summarize, this new experimental procedure that allows simultaneous monitoring of IDSR and vis−vis−UV holeburning conformation-specific spectra can serve as an important, indicative means for identification of structures and for studying dynamics of isolated biomolecules. It is significant that vis−vis−UV hole burning may take place while measuring IDSR spectra and that the former could be distinguished from the latter by their very different line widths. This method can be foreseen to work for the other conformers of TRA, as well as for other molecules, provided that the ωp and ωS beams are chosen with wavelengths that can sample the upper potential energy surface.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The support of this research by the Israel Science Foundation (ISF) and by the James Franck Binational German−Israeli Program in Laser−Matter Interaction is gratefully acknowledged.

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