The dynamics of conformational isomerization are explored in a methyl-capped dipeptide, N-acetyl-tryptophan methyl amide (NATMA), using infrared-ultraviolet (IR-UV) hole-filling and IR-induced population transfer spectroscopies. IR radiation selectively excites individual NH stretch vibrational fundamentals of single conformations of the molecule in the early portions of a gas-phase expansion, and then this excited population is collisionally recooled into its conformational minima for subsequent conformation-specific detection. Efficient isomerization is induced by the IR excitation that redistributes population between the same conformations that have population in the absence of IR excitation. The quantum yields for transfer of the population into the various conformational minima depend uniquely on which conformation is excited and on which NH stretch vibration is excited within a given conformation.
A combination of methods, including laser-induced fluorescence excitation, fluorescence-dip infrared ͑FDIR͒ spectroscopy, and UV-UV hole-burning spectroscopy, have been used to study the infrared and ultraviolet spectra of single conformations of two methyl-capped dipeptides: N-acetyl tryptophan amide ͑NATA͒ and N-acetyl tryptophan methyl amide ͑NATMA͒. Density functional theory calculations predict that all low-energy conformers of NATA and NATMA belong to one of two conformational families: C5, with its extended dipeptide backbone, or C7 eq , in which the dipeptide backbone forms a seven-membered ring joined by a H bond between the-amide NH and the-amide carbonyl groups. In NATA ͑NATMA͒, the LIF spectrum has contributions from two ͑three͒ conformers. FDIR spectroscopy has been used to record infrared spectra of the individual conformers over the 2800-3600 cm Ϫ1 region, free from interference from one another. The NH stretch region provides unequivocal evidence that one of the conformers of NATA is C5, while the other is C7 eq. Similarly, in NATMA, there are two C5 conformers, and one C7 eq structure. Several pieces of evidence are used to assign spectra to particular C5 and C7 eq conformers. NATA͑A͒ and NATMA͑B͒ are both assigned as C5͑AP͒ structures, NATA͑B͒ and NATMA͑C͒ are assigned as C7 eq ͑⌽P͒, and NATMA͑A͒ is assigned as C5͑A⌽͒. In both molecules, the C5 structures have sharp vibronic spectra, while the C7 eq conformers are characterized by a dense, highly congested spectrum involving long progressions that extend several hundred wave numbers to the red of the C5 S 1-S 0 origins. N-acetyl tryptophan ethyl ester ͑NATE͒, which can only form C5 conformers, shows only sharp transitions in its LIF spectrum due to four C5 conformers, with no evidence for the broad absorption due to C7 eq. This provides direct experimental evidence for the importance of the peptide backbone conformation in controlling the spectroscopic and photophysical properties of tryptophan.
The electronic and infrared spectra of anthranilic acid in a supersonic jet were measured. The fluorescence excitation spectrum is extremely congested. IR-UV hole-burning measurements indicate that only a single ground-state rotamer contributes to the observed spectrum. Vibrational progressions in 252 and 418 cm -1 modes were observed. Density functional theory calculations indicate that these modes involve the in-plane bending motions of the amino and carboxyl groups of anthranilic acid. The presence of vibrational progressions in these modes suggests that the relative positions of the amino and carboxyl groups are different in the ground and excited electronic states of anthranilic acid. This observation is supported by the fluorescence-dip infrared spectra acquired, which show a shift in the lower frequency NH stretch fundamental from 3394 to 2900 cm -1 upon electronic excitation, suggesting a dramatic strengthening of the intramolecular hydrogen bond in the excited state. The change in the hydrogen-bond strength does not lead to full excited-state intramolecular hydrogen-atom transfer, as the strongly red-shifted emission feature associated with this process is not observed. Instead, the excited-state behavior of anthranilic acid is best described as intramolecular hydrogen-atom dislocation, as has been postulated for the related molecule salicylic acid.
The conformational isomerization dynamics of N-acetyl tryptophan methyl amide (NATMA) and N-acetyl tryptophan amide (NATA) have been studied using the methods of IR-UV hole-filling spectroscopy (HFS) and IR-induced population transfer spectroscopy (IR-PTS), which were developed for this purpose. Single conformations of these molecules were selectively excited in well-defined NH stretch fundamentals. This excess energy was used to drive conformational isomerization. By carrying out the infrared excitation early in a supersonic expansion, the excited molecules were recooled into their zero-point levels, partially refilling the hole created in the ground state population of one of the conformers, and creating gains in population in other conformers. These changes in population were detected using laser-induced fluorescence downstream in the expansion. In HFS, the IR wavelength is fixed and the UV laser tuned in order to determine where the population went following selective infrared excitation. In IR-PTS, the UV is fixed to monitor the population of a given conformation, and the IR is tuned to record the IR-induced changes in the population of the monitored conformer. Besides demonstrating the capability of the experiment to change the downstream conformational population distribution, the IR-PTS scans were used to extract two quantitative results: (i) The fractional populations of the conformers in the absence of the infrared, and (ii) the isomerization quantum yields for each of the six unique amide NH stretch fundamentals (three conformers each with two amide groups). The method for obtaining quantum yields is described in detail. In both NATMA and NATA, the quantum yields show modest conformational specificity, but only a hint of vibrational mode specificity. The prospects for the hole-filling technique for providing insight into energy flow in large molecules are discussed, leaving a more detailed theoretical modeling to the adjoining paper [Evans et al. J. Chem. Phys. 120, 148 (2004)].
The hydride stretch infrared spectra of indole, indole-H 2 O, 3-methyl indole, 3-methyl indole-H 2 O, the main conformer of tryptamine ͑TRA͒, two conformers of N-acetyl tryptophan amide ͑NATA͒, and three conformers of N-acetyl tryptophan methyl amide ͑NATMA͒, have been recorded in the electronically excited singlet states using excited-state fluorescence-dip infrared spectroscopy. NATA and NATMA are methyl-capped dipeptides of tryptophan that have conformational flexibility and exhibit sensitivity in their electronic spectra to the conformation of the dipeptide backbone. In the indole monomer, the indole NH stretch fundamental at the S 1 origin is shifted from its ground-state value ͑3525 cm Ϫ1 ͒ to 3478 cm Ϫ1 . The corresponding band in the indole-H 2 O complex appears at 3387 cm Ϫ1 , shifted by a similar amount from its ground-state position ͑3436 cm Ϫ1 ͒. Higher vibronic levels within 1500 cm Ϫ1 of the S 1 origin, which have been identified previously ͓B. J. Fender et al., Chem. Phys. Lett. 239, 31 ͑1995͔͒ as being 1 L b or 1 L a in character, all show similar excited state indole NH stretch absorptions. The corresponding spectra in 3-methyl indole, 3-methyl indole-H 2 O, TRA, and in the C5 conformers of NATA and NATMA all are missing the indole NH stretch absorption. In its place, a broad background absorption appears, spread over the entire 2800-3800 cm Ϫ1 region. In these molecules, other CH stretch or amide NH stretch absorptions remain sharp, appearing in their expected frequency ranges. Finally, the C7 conformations of NATA and NATMA, which possess an intramolecular hydrogen bond in the dipeptide backbone, have all infrared transitions washed out, replaced by a stronger broad background absorption. The entire data set can be explained by the presence of an excited 1 * state which is dissociative along the indole NH stretch coordinate, as recently predicted by Sobolewski and Domcke ͓Chem. Phys. Lett. 315, 293 ͑1999͔͒. In the weak coupling case ͑indole, indole-H 2 O), the gap between the 1 * state and the S 1 origin is too large to be reached by infrared excitation. The selective loss of the indole NH stretch in the intermediate coupling case reflects the strong coupling of the 1 L b state NH stretch (vϭ1) level to the 1 * state, which is dissociative along the NH stretch coordinate. In the NATA and NATMA C7 conformers, an inversion of ordering of the electronic states occurs, pushing the 1 L a state below the 1 L b origin, and strengthening the coupling of all hydride stretch vibrational levels to the 1 * dissociative continuum. These results highlight the important influence of the conformation of the polypeptide backbone on the photophysics of tryptophan in polypeptides.
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