Experimental determination of conformational isomerization energy thresholds in serotonin.

Stimulated emission pumping-population transfer (SEP-PT) and hole-filling (SEP-HF) spectroscopies were used to determine the energy thresholds to isomerization between thirteen reactant-product conformer pairs in the biomolecule serotonin (SERO). Serotonin is a close structural analog of tryptamine (TRA), differing in having a hydroxyl group in the 5 position of the indole ring. A previous spectroscopic study (LeGreve; et al. J. Am. Chem. Soc. 2007, 129 (13), 4028) identified eight conformational isomers of SERO, whose interconversion involves motion of the 3-ethylamine side chain, the 5-OH group, or both. In the cases in which only an ethylamine side chain reorientation occurred, the barriers were found to be similar to, but systematically somewhat smaller than, those in TRA, which has been studied by similar methods (Dian; et al. Science 2004, 303 (5661), 1169; Clarkson; et al. J. Chem. Phys. 2005, 122 (21), Art. No. 214311). In most cases, the experimental thresholds are well reproduced by calculated transition states separating the conformational wells; however, tunneling effects may artificially reduce the thresholds observed for isomerization of SERO(A,Gpy(out)) and SERO(B,Gpy(up)) into SERO(C,Gph(out)). The A --> A' isomerization involving only the OH rotation from anti to syn was found to be 721-761 cm-1, in accordance with the calculated classical barrier. For isomerizations in which the ethylamine side chain reorients as does the OH group, the barriers to isomerization were consistent with sequential rather than concerted motion of both groups. Finally, some evidence for mode-specific effects in the product quantum yields near threshold is presented.

Entropy-driven population distributions in a prototypical molecule with two flexible side chains: O-(2-acetamidoethyl)-N-acetyltyramine.

Resonant two-photon ionization (R2PI), resonant ion-dip infrared (RIDIR), and UV-UV hole-burning spectroscopies have been employed to obtain conformation-specific infrared and ultraviolet spectra under supersonic expansion conditions for O-(2-acetamidoethyl)-N-acetyltyramine (OANAT), a doubly substituted aromatic in which amide-containing alkyl and alkoxy side chains are located in para positions on a phenyl ring. For comparison, three single-chain analogs were also studied: (i) N-phenethyl-acetamide (NPEA), (ii) N-(p-methoxyphenethyl-acetamide) (NMPEA), and (iii) N-(2-phenoxyethyl)-acetamide (NPOEA). Six conformations of OANAT have been resolved, with S(0)-S(1) origins ranging from 34,536 to 35,711 cm(-1), denoted A-F, respectively. RIDIR spectra show that conformers A-C each possess an intense, broadened amide NH stretch fundamental shifted below 3400 cm(-1), indicative of the presence of an interchain H bond, while conformers D-F have both amide NH stretch fundamentals in the 3480-3495 cm(-1) region, consistent with independent-chain structures with two free NH groups. NPEA has a single conformer with S(0)-S(1) origin at 37,618 cm(-1). NMPEA has three conformers, two that dominate the R2P1 spectrum, with origin transitions between 35,580 and 35,632 cm(-1). Four conformations, one dominate and three minor, of NPOEA have been resolved with origins between 35,654 and 36,423 cm(-1). To aid the making of conformational assignments, the geometries of low-lying structures of all four molecules have been optimized and the associated harmonic vibrational frequencies calculated using density functional theory (DFT) and RIMP2 methods. The S(0)-S(1) adiabatic excitation energies have been calculated using the RICC2 method and vertical excitation energies using single-point time-dependent DFT. The sensitivity of the S(0)-S(1) energy separation in OANAT and NPOEA primarily arises from different orientations of the chain attached to the phenoxy group. Using the results of the single-chain analogs, tentative assignments have been made for the observed conformers of OANAT. The RIMP2 calculations predict that interchain H-bonded conformers of OANAT are 25-30 kJ/mol more stable than the extended-chain structures. However, the free energies of the interchain H-bonded and extended structures calculated at the preexpansion temperature (450 K) differ by less than 10 kJ/mol, and the number of extended structures far outweighs the number of H-bonded conformers. This entropy-driven effect explains the presence of the independent-chain conformers in the expansion, and cautions future studies that rely solely on relative energies of conformers in considering possible assignments.

Infrared photodissociation of a water molecule from a flexible molecule-H2O complex: rates and conformational product yields following XH stretch excitation.

Infrared-ultraviolet hole-burning and hole-filling spectroscopies have been used to study IR-induced dissociation of the tryptamine.H2O and tryptamine.D2O complexes. Upon complexation of a single water molecule, the seven conformational isomers of tryptamine collapse to a single structure that retains the same ethylamine side chain conformation present in the most highly populated conformer of tryptamine monomer. Infrared excitation of the tryptamine.H2O complex was carried out using a series of infrared absorptions spanning the range of 2470-3715 cm-1. The authors have determined the conformational product yield over this range and the dissociation rate near threshold, where it is slow enough to be measured by our methods. The observed threshold for dissociation occurred at 2872 cm-1 in tryptamine.H2O and at 2869 cm-1 in tryptamine.D2O, with no dissociation occurring on the time scale of the experiment (approximately 2 micros) at 2745 cm-1. The dissociation time constants varied from approximately 200 ns for the 2869 cm-1 band of tryptamine.D2O to approximately 25 ns for the 2872 cm-1 band of tryptamine.H2O. This large isotope dependence is associated with a zero-point energy effect that increases the binding energy of the deuterated complex by approximately 190 cm-1, thereby reducing the excess energy available at the same excitation energy. At all higher energies, the dissociation lifetime was shorter than the pulse duration of our lasers (8 ns). At all wavelengths, the observed products in the presence of collisions are dominated by conformers A and B of tryptamine monomer, with small contributions from the other minor conformers. In addition, right at threshold (2869 cm-1), tryptamine.D2O dissociates exclusively to conformer A in the absence of collisions with helium, while both A and B conformational products are observed in the presence of collisions with helium. Using resolution-of-identity approximation to second-order Moller-Plesset binding energies extrapolated to the complete basis set limit and harmonic vibrational frequencies and transition states calculated at the density functional limit B3LYP/6-31+G* level of theory, Rice-Ramsperger-Kassel-Marcus (RRKM) predictions for the dissociation, isomerization, and water shuttling rates as a function of energy are made. At threshold, the experimental dissociation rate is almost 10(3) faster than RRKM predictions. Reasons for this apparent non-RRKM behavior will be discussed.

Direct measurement of the energy thresholds to conformational isomerization in tryptamine: experiment and theory.

The methods of stimulated emission pumping-hole filling spectroscopy (SEP-HFS) and stimulated emission pumping population transfer spectroscopy (SEP-PTS) were applied to the conformation-specific study of conformational isomerization in tryptamine [TRA, 3-(2-aminoethyl)indole]. These experimental methods employ stimulated emission pumping to selectively excite a fraction of the population of a single conformation of TRA to well-defined ground-state vibrational levels. This produces single conformations with well-defined internal energy, tunable over a range of energies from near the zero-point level to well above the lowest barriers to conformational isomerization. When the SEP step overcomes a barrier to isomerization, a fraction of the excited population isomerizes to form that product. By carrying out SEP excitation early in a supersonic expansion, these product molecules are subsequently cooled to their zero-point vibrational levels, where they can be detected downstream with a third tunable laser that probes the ground-state population of a particular product conformer via a unique ultraviolet transition using laser-induced fluorescence. The population transfer spectra (recorded by tuning the SEP dump laser while holding the pump and probe lasers fixed) exhibit sharp onsets that directly determine the energy thresholds for conformational isomerization in a given reactant-product conformer pair. In the absence of tunneling effects, the first observed transition in a given X-Y PTS constitutes an upper bound to the energy barrier to conformational isomerization, while the last transition not observed constitutes a lower bound. The bounds for isomerizing conformer A of tryptamine to B(688-748 cm(-1)), C(1)(860-1000 cm(-1)), C(2)(1219-1316 cm(-1)), D(1219-1282 cm(-1)), E(1219-1316 cm(-1)), and F(688-748 cm(-1)) are determined. In addition, thresholds for isomerizing from B to A(<1562 cm(-1)), B to F(562-688 cm(-1)), and out of C(2) to B(<747 cm(-1)) are also determined. The A-->B and B-->A transitions are used to place bounds on the relative energies of minima B relative to A, with B lying at least 126 cm(-1) above A. The corresponding barriers have been computed using both density functional and second-order many-body perturbation theory methods in order to establish the level of theory needed to reproduce experimental results. While most of the computed barriers match experiment well, the barriers for the A-F and B-F transitions are too high by almost a factor of 2. Possible reasons for this discrepancy are discussed.

Direct measurement of the energy thresholds to conformational isomerization. II. 3-indole-propionic acid and its water-containing complex.

The methods of stimulated emission pumping-hole-filling spectroscopy (SEP-HFS) and population transfer spectroscopy (SEP-PTS) were used to place direct experimental bounds on the energetic barriers to conformational isomerization in 3-indole-propionic acid (IPA) and its water-containing complex. By contrast with tryptamine (Paper I), IPA has only two conformations with significant population in them. The structures of the two conformers are known from previous work [P. M. Felker, J. Phys. Chem. 96, 7844 (1992)]. The energy thresholds for A-->B and B-->A isomerizations are placed at 854 and 754 cm(-1), respectively. Lower bounds on the isomerization barrier in the two directions are determined from the last transition not observed in the SEP-PT spectra. These are placed at 800 and 644 cm(-1) for A-->B and B-->A, respectively. The combined results place bounds on the relative energies of the A and B minima, with E(B)-E(A)=46-210 cm(-1). Like the IPA monomer, the IPA-H2O complex forms two conformational isomers. Both these isomers incorporate the water molecule as a bridge between the carbonyl and OH groups of the carboxylic acid. Previous rotational coherence measurements (L. L. Connell, Ph.D. thesis, UCLA, 1991) have determined that these complexes retain the same IPA conformational structure as the monomers. SEP-PTS and SEP-HFS were carried out on the IPA-H2O complexes. It was demonstrated that it is possible to use SEP to drive conformational isomerization between the two conformational isomers of IPA-H2O. Bounds on the energy barriers to conformational isomerization are not effected greatly by the presence of the water molecule, with Ebarrier(A-->B)=771-830 cm(-1) and Ebarrier(B-->A)=583-750 cm(-1). This is a simple consequence of the fact that the barrier is an intramolecular barrier, and the water molecule is held fixed in the COOH pocket, where it interacts with the ring only peripherally during the isomerization process. Finally, changes in the SEP-PT spectral intensity in transitions near the top of the barrier to isomerization as a function of the position of SEP excitation relative to the pulsed valve exit provide some insight to the competition between vibrational relaxation and isomerization in a molecule the size of IPA.

Laser-initiated shuttling of a water molecule between H-bonding sites.

The two-step laser excitation scheme of stimulated emission pumping (SEP) induces shifts of a single water molecule between two remote hydrogen bonding sites on trans-formanilide. This reaction can be initiated by selective excitation of either isomer (CO-bound or NH-bound) with different SEP excitation wavelengths. Energy (E) thresholds for isomerization in both directions have been measured [796 wave numbers NH) CO)

Isomer-specific spectroscopy and conformational isomerization energetics of o-, m-, and p-ethynylstyrenes.

The infrared and ultraviolet spectroscopy of o-, m-, and p-ethynylstyrene isomers (oES, mES, and pES) were studied by a combination of methods, including resonance-enhanced two-photon ionization (R2PI), UV-UV hole-burning spectroscopy (UVHB), resonant ion-dip infrared spectroscopy (RIDIRS), and rotationally resolved fluorescence excitation spectroscopy. In addition, the newly developed method of stimulated emission pumping-population transfer spectroscopy (SEP-PTS) was used to determine the energy threshold to conformational isomerization in m-ethynylstyrene. The S(1) <-- S(0) origin transitions of oES and pES occur at 32 369 and 33 407 cm(-1), respectively. In mES, the cis and trans conformations are calculated to be close in energy. In the R2PI spectrum of mES, the two most prominent peaks (32672 and 32926 cm(-1)) were confirmed by UVHB spectroscopy to be S(1) <-- S(0) origins of these two conformers. The red-shifted conformer was identified as the cis structure by least-squares fitting of the rotationally resolved fluorescence excitation spectrum of the origin band. There are also two possible conformations in oES, but transitions due to only one were observed experimentally, as confirmed by UVHB spectroscopy. Density functional theory calculations (B3LYP/6-31+G) predict that the cis-ortho conformer, in which the substituents point toward each other, is about 8 kJ/mol higher in energy than the trans-ortho isomer, and should only be about 5% of the room temperature population of oES. Ground-state infrared spectra in the C-H stretch region (3000-3300 cm(-1)) of each isomer were obtained with RIDIRS. In all three structural isomers, the acetylenic C-H stretch fundamental was split by Fermi resonance. Infrared spectra were also recorded in the excited electronic state, using a UV-IR-UV version of RIDIR spectroscopy. In all three isomers the acetylenic C-H stretch fundamental was unshifted from the ground state, but no Fermi resonance was seen. The first observed and last unobserved transitions in the SEP-PT spectrum were used to place lower and upper bounds on the barrier to cis --> trans isomerization in m-ethynylstyrene of 990-1070 cm(-1). Arguments are given for the lack of a kinetic shift in the measurement. The analogous trans --> cis barrier is in the same range (989-1065 cm(-1)), indicating that the relative energies of the zero-point levels of the two isomers are (E(ZPL)(cis) - E(ZPL)(trans))= -75 to +81 cm(-1). Both the barrier heights and relative energies of the minima are close to those determined by DFT (Becke3LYP/6-31+G) calculations.

Infrared-induced conformational isomerization and vibrational relaxation dynamics in melatonin and 5-methoxy-N-acetyl tryptophan methyl amide.

The conformational isomerization dynamics of melatonin and 5-methoxy N-acetyltryptophan methyl amide (5-methoxy NATMA) have been studied using the methods of IR-UV hole-filling spectroscopy and IR-induced population transfer spectroscopy. Using these techniques, single conformers of melatonin were excited via a well-defined NH stretch fundamental with an IR pump laser. This excess energy was used to drive conformational isomerization. By carrying out the infrared excitation early in a supersonic expansion, the excited molecules were re-cooled into their zero-point levels, partially re-filling the hole created in the ground state population of the excited conformer, and creating gains in population of the other conformers. These changes in population were detected using laser-induced fluorescence downstream in the expansion via an UV probe laser. The isomerization quantum yields for melatonin show some conformation specificity but no hint of vibrational mode specificity. In 5-methoxy NATMA, no isomerization was observed out of the single conformational well populated in the expansion in the absence of the infrared excitation. In order to study the dependence of the isomerization on the cooling rate, the experimental arrangement was modified so that faster cooling conditions could be studied. In this arrangement, the pump and probe lasers were overlapped in space in the high density region of the expansion, and the time dependence of the zero-point level populations of the conformers was probed following selective excitation of a single conformation. The analysis needed to extract isomerization quantum yields from the timing scans was developed and applied to the melatonin timing scans. Comparison between the frequency and time domain isomerization quantum yields under identical experimental conditions produced similar results. Under fast cooling conditions, the product quantum yields were shifted from their values under standard conditions. The results for melatonin are compared with those for N-acetyl tryptophan methyl amide.

Direct measurement of energy thresholds to conformational isomerization in tryptamine.

Stimulated emission pumping (SEP)-hole filling spectroscopy and SEP-induced population transfer spectroscopy have been used to place narrow bounds on the energy thresholds for isomerization between individual reactant-product isomer pairs involving the seven conformational minima of tryptamine. The thresholds for isomerizing conformer A to all six other conformations divided into three groups at 750 wavenumbers (cm-1)(A-->B, F), 1000 cm-1 [A-->C(2)], and 1280 to 1320 cm-1 [A-->D, E, and C(1)]. The appearance of the first band and the absence of the band below it were used to place upper and lower bounds to the barrier heights for each process. The thresholds for A-->B and B-->A isomerizations were also combined to determine the relative energies of these two lowest energy minima. The combined data from all X-->Y isomerizations identify important isomerization pathways on the potential energy surface linking the minima.