Hiding in Plain Sight: Unmasking the Diffuse Spectral Signatures of

Letter pubs.acs.org/JPCL

Hiding in Plain Sight: Unmasking the Diffuse Spectral Signatures of the Protonated N‑Terminus in Isolated Dipeptides Cooled in a Cryogenic Ion Trap Christopher M. Leavitt,†,§ Andrew F. DeBlase,† Christopher J. Johnson,† Michael van Stipdonk,# Anne B. McCoy,‡ and Mark A. Johnson*,† †

Sterling Chemistry Laboratory, Yale University, New Haven, Connecticut 06520, United States Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15285, United States ‡ Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States #

S Supporting Information *

ABSTRACT: Survey vibrational predissociation spectra of several representative protonated peptides and model compounds reveal very diffuse absorptions near 2500 cm−1 that are traced to pentagonal cyclic ionic hydrogen bonds (C5 interactions) involving the excess charge centers. This broadening occurs despite the fact that the ions are cooled close to their vibrational zero-point levels and their spectra are obtained by predissociation of weakly bound adducts (H2, N2, CO2) prepared in a cryogenic ion trap. The C5 band assignments are based on H/D isotopic substitution, chemical derivatization, solvation behavior, and calculated spectra. We evaluate the extent to which this broadening is caused by anharmonic coupling in the isolated molecules by including cubic coupling terms in the normal mode expansion of the potential energy surface. This analysis indicates that the harmonic H-bonded stretching vibration is mixed with dark background states over much of the energy range covered by the observed features. The difficulty with identifying these features in earlier studies of dipeptides is traced to both the breadth and the fact they are calculated to be intrinsically weaker than cases involving linear variations of the N···H+···O motif. SECTION: Spectroscopy, Photochemistry, and Excited States

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size C(5+3n), where n is the number of residues removed from the N-terminus. For example, infrared spectra of vibrationally cold Ac-Phe-(Ala)n=5,10-LysH+ ions,19,30,31 acquired using an IR−UV double resonance technique, supported previous ion mobility studies32 that identified formation of helical structures. Most often, however, such spectra are obtained using infrared multiple photon dissociation (IRMPD) of room-temperature ions,20,33−41 and the resulting broad bands are not readily assigned to particular structures. Rather, finite temperature simulations39,42−45 of such spectra indicate families of structures that contribute to the various features. Here, we are particularly interested in the spectral signature of the embedded proton in simple dipeptides, such as GlyGlyH+, for which the most stable structure is calculated to occur with the pentagonal or C5 H-bonding (C5 HB) motif displayed in Scheme 1. In that arrangement, protonation at the N-terminus results in an NH that folds back onto a nearby CO in the same residue.19,20,40,41,46

nderstanding the extreme variations in the spectral behavior associated with vibrations of an excess proton embedded in a scaffold of heavy atoms remains a profound challenge for contemporary molecular spectroscopy.1−19 On one hand, protons added to a lone pair of electrons on isolated secondary amines or alcohols appear in the spectrum as sharp bands arising from the collective normal modes associated with the identical (XH) oscillators in the ROH2+ and R2NH2+ groups. When these groups are brought into the proximity of a H-bond acceptor, however, one of the two XH groups becomes engaged in a very strong inter- or intramolecular ionic hydrogen bond (IHB). The vibrational signatures of the engaged NH or OH stretches are red-shifted by hundreds to thousands of wavenumbers, while the sharp nonbonded XH transitions are largely unaffected. In many cases, these strongly red-shifted bands can be readily identified because they are very intense and exhibit appropriate shifts upon H/D substitution at the bridging site.18 The present study was in part motivated by the recent flurry of activity directed toward exploiting vibrational spectroscopy to identify the fundamental interactions that dictate secondary structure in proteins and peptides isolated in the gas phase.6,20−30 In larger peptides intramolecular H-bonds formed with CO groups along the backbone result in rings of the © 2013 American Chemical Society

Received: August 7, 2013 Accepted: September 20, 2013 Published: September 20, 2013 3450

dx.doi.org/10.1021/jz401681y | J. Phys. Chem. Lett. 2013, 4, 3450−3457

The Journal of Physical Chemistry Letters

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Scheme 1. Illustration of the C5 Hydrogen-Bonding Motif in GlyGlyH+ along with Selected Structural Parameters to Illustrate the Asymmetry of the Linkage

The spectral signatures of the C5 motif have been addressed in previous work, such as the simulations of the 300 K IRMPD spectrum of Ala2H+ by Cimas et al.42 These authors suggest that the internal H-bond contributes to activity over the entire range from 2500 to 2850 cm−1, where the large red-shift was rationalized in the context of the anharmonic potential for the bridging proton displacement between the H-bond donor and acceptor atoms while the width was attributed to the dynamics of the H-bond at finite temperature, including migration of the proton onto the proximal CO group. It is thus of interest to establish the intrinsic behavior of this C5 motif, i.e., that displayed by vibrationally cold ions using a linear spectroscopic approach to facilitate the comparison with theory. Interestingly, several such studies have appeared, but in fact have only added to the confusion! In one case, the cryogenic vibrational bands observed for small protonated peptides46−48 using H2 tagging could all be accounted for without including the H-bond linkages. A similar anomaly regarding the “missing” C5 HB NH stretch was also recently reported for protonated TyrAla by Stearns et al.,19 where the NH engaged in the C5 (NH···OC) motif was the only NH oscillator that could not be identified in the IR−UV spectra of the vibrationally cold, bare ion. This led them to conclude that the IHB feature must be so broadened that its peak absorption is much weaker than those of the sharper fundamentals. In this Letter, the vibrational predissociation spectra of three cryogenically cooled dipeptide derivatives are presented in an effort to reveal the locations and breadths of the bands attributed to these C5 linkages. The key C5 HB absorptions are identified by following their response to H/D isotopic substitution and confirmed by their solvent shifts when one of the nonbonded NH groups on the N-terminus is complexed with a weak base (R in Scheme 1), which effectively weakens the intramolecular H-bond. Our previous reports of the vibrational predissociation spectra of the H2-tagged, protonated SarGlyH+ and GlyGlyH+ had a break in the spectral coverage from 1800 to 2500 cm−1 due to a changeover in laser configurations that resulted in a dramatic reduction in laser power in that range. This is unfortunate because the critical bands predicted for the C5 HB are calculated to be red-shifted close to this gap, as indicated by the harmonic spectrum for GlyGlyH+ in Figure 1b. In those studies, the only band observed in the restricted scan range that could not be readily assigned at the harmonic level for either GlyGlyH+ or GlySarH+ was a broadened featured at 1350 cm−1 (* in Figure 1a). Consequently, this feature was tentatively assigned to C5 HB activity,46,47 and the assignment was rationalized by a strongly anharmonic potential for the H+ stretching vibration between the N-atom donor and O-atom

Figure 1. (a) Vibrational predissociation spectrum of GlyGlyH+·2H2 and (b) calculated harmonic spectrum (B3LYP/6-311++G(d,p)) for the global minimum structure indicated at the top. The harmonic frequency spectrum was scaled by a factor of 0.987 to bring the predicted acid CO stretch into agreement with the experimental band. The band previously identified with the bridging proton activity is indicated by *, while fundamentals associated with various motions of the scaffold are noted below. The red arrow marks the week feature centered at 2490 cm−1, which is now (vida infra) the candidate for the signature of the C5 HB interaction, while the band labeled with a * was previously given a tentative assignment to the C5 HB active transition because of its absence at the harmonic level.

acceptor of the intramolecular H-bond. More recently, however, we noted in a Letter48 describing three different cyclic isomers of the closely related SarSarH+ peptide that the conformer with the same C5 HB motif as that shown in Scheme 1 exhibited a very broad (200 cm−1) feature near 2700 cm−1 that was clearly assigned as the H-bonded NH stretch of the protonated N-terminus. To clarify the assignments, we reacquired the spectra of several model dipeptide compounds with Gly at the N-terminus using a laser system that was optimized to provide sufficient photon flux in the gap to allow acquisition of the entire spectral range. The resulting spectrum of GlyGlyH+ is presented in Figure 1, along with the calculated harmonic vibrational spectrum and the minimum-energy structure.46 Note that the complete scan obtained here does not reveal any sharp or strong features in the 1800−2500 cm−1 range, although the increased signal-to-noise available with the current instrument reveals a broad and weak absorption (red arrow in the trace in Figure 1a) centered at ∼2490 cm−1 that was not evident in the earlier scan. Figure 2 displays the vibrational predissociation spectra of several H2-tagged, protonated dipeptide model compounds to explore whether the broad band recovered in GlyGlyH+ (red arrow in Figure 1a) is a general property of these species. The upper three traces correspond to nicotinyl-glycine (NicGlyH+), sarcosyl-glycine (SarGlyH+), and GlyGlyH+ in Figure 2a−c, respectively, with the structures indicated in the insets. The lower traces (Figure 2d and e) compare the experimental and (scaled) harmonic spectra of the D5-deuterated isotopologue of GlyGlyH+ (i.e., deuteration of all NH and OH hydrogen atoms). The all-H isotopologues display sharp free OH stretching bands at 3567 cm−1 arising from the acid groups that, in these cases, are not involved in H-bonding. About 200 cm−1 lower in energy are similarly sharp bands derived 3451



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quite similar to that of NicGlyH+ (Figure 2a). Closer inspection, however, reveals that both spectra display broad, weak features (highlighted red in Figure 2b and c and expanded ×8 in the insets). Importantly, the centroid of the SarGlyH+ absorption is blue-shifted relative to that in GlyGlyH+ by roughly the same amount predicted at the harmonic level (red arrows in Figure 2b and c) for the C5 HBs in both systems. In each case, the diffuse feature is observed about 200 cm−1 below the harmonic prediction for the NH group involved in the C5 HB, which is the only harmonic fundamental in the entire range between 2000 and 3000 cm−1. Note that all other features, higher and lower in energy, are relatively sharp and accurately predicted (with typical scaling) at the harmonic level for the GlyGlyH+ minimum-energy structure, which is displayed at the right of Figure 2e. An obvious way to challenge the assignment of the broad feature at around 2500 cm−1 to the C5 HB signature is to monitor the evolution of the band pattern upon deuteration. Because there are several exchangeable hydrogen atoms, we focus on the isotopologue with D in all exchangeable sites (denoted GlyGlyD+). Its spectrum is presented in Figure 2d with the scaled (see caption) harmonic prediction for the structure at the right of the trace in Figure 2e displayed inverted below it. This emphasizes that all bands are indeed well described at the harmonic level, with the exception of the C5 DB band centered at 1920 cm−1, which is calculated at the harmonic level to occur at 2046 cm−1. Once again, a diffuse, weak band occurs in the experimental spectrum (highlighted red) peaking at about 170 cm−1 below the harmonic value. This behavior suggests that the broad bands highlighted red in Figure 2 are, in fact, derived from the NH(D) groups involved in C5 HBs such that the intensity expected at the harmonic level is distributed among a large number of otherwise low-oscillator-strength transitions spread over a large energy range. Indeed, the integrated intensity of the red feature in the spectrum of GlyGlyH+, relative to that of the observed free OH stretch, is in close agreement with the predicted ratio in the harmonic fundamentals (4.5 versus 3.9 from experiment and B3LYP/6-311++G(d,p) calculations, respectively). We note in passing that this ratio is much smaller than that displayed by the linear NH+···O analogues (NH4+·OH2) with a similar red-shift (724 versus 870 cm−1 for NH4+·OH2 and GlyGlyH+, respectively).18 In the linear complex, the observed IHB intensity relative to the H2O symmetric OH stretch is about 30, which is very close to the harmonic prediction of 29. The facts that the key H-bonded bands in the linear complexes are both more intense and narrower (by more than a factor or two) thus allowed them to be readily identified in those cases but missed in earlier analyses of the cyclic variations. A comparison between the NH4+·OH2 and GlyGlyH + spectra is included in the Supporting Information (Figure S1). Having provisionally identified the C5 HB signatures, it is useful to explore how they respond to systematic changes in both scaffold and solvation. If we regard the general location of the band as a reflection of the competition between the proton affinities (PAs) of the two H-bond acceptors, R1R2N and CO, then the band should shift according to the systematic changes in the basicity of the N-atom.18,49 One way to achieve this is by alkyl substitution at the N-terminus (i.e., Ri = CH3 versus H), where methylation draws the bridging proton closer to the N-atom, thereby strengthening the NH-bond and weakening the cyclic H-bond. This effect was extensively

Figure 2. Vibrational predissociation spectra of (a) NicGlyH+·(D2)2, (b) SarGlyH+·(H2)2, and (c) GlyGlyH+·(H2)2 collected by monitoring the photoinduced dissociation of both H2/D2 molecules. The vibrational spectrum of the D5-GlyGlyH+ isotopologue, (i.e., all OH and NH groups exchanged for deuterium) is presented in trace (d), with the corresponding harmonic spectrum (B3LYP/6-311++G(d,p)) presented inverted in (e). The calculated spectrum was scaled by 0.987 to bring the predicted acid CO stretch into agreement with the experimental band. Arrows in (b) and (c) indicate the harmonic predictions for the locations of the NH stretches involved in the C5 hydrogen bonds (C5 HB), while bands highlighted in red are assigned to absorptions derived from this motion. The blue/red gradient colored arrow depicts the red shift in the NH fundamental as it engages in H-bonding. The wireframe structure on the lower right corresponds to the minimum-energy geometry of GlyGlyH+. The expanded scale in (d) highlights the fact the CH stretching bands are very weak and do not contribute to the C5 HB activity in this region.

from the amide NH and nonbonded NH groups at the Nterminus. As has been previously noted in the spectrum of GlyGlyH+,46,47 the free NH stretches of the protonated amine are red-shifted by ∼10 cm−1 upon complexation with additional H2 tags (see below), resulting in the asymmetric NH2 and embedded amide NH stretches observed as a single band centered at 3360 cm−1 in the spectrum of GlyGlyH+·(H2)2. The NicGlyH+ system (trace in Figure 2a) was chosen because the N-terminal residue features a pyridyl group that forces the excess proton onto a remote site where it cannot engage in an intramolecular H-bond with any of the other basic sites in the molecule. As such, its spectrum would reveal any residual spectral activity in the critical 2000−3000 cm−1 region that is not associated with a C5 linkage. This region is completely clear, however, in the NicGlyH+ spectrum, which displays only sharp transitions from the two NH groups near 3400 cm−1 as well as the weaker CH stretches around 3000 cm−1. The spectra of SarGlyH+ and GlyGlyH+ are presented in Figure 2b and c, respectively, and at first glance, they appear 3452



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motifs were identified based on the vibrational spectra. One class of isomers contained a free amino NH and a single C5 linkage, where the other conformer formed an additional Hbond between the formerly free amine NH and the acid CO, thus enabling a detailed study of how the C5 HB bands evolve when the free NH (3331 cm−1) binds to another site within the molecule. The single H-bonded structure yielded a broad C5 HB feature near 2700 cm−1 that blue-shifted with a concomitant red-shift of the initially free NH stretch, resulting in two nearby bands at ∼3120 and 3200 cm−1 from the embedded −NH2 group when both NHs were engaged in cyclic intramolecular H-bonds. One aspect of the solvation behavior of GlyGlyH+ that is not obvious is that the C5 HB feature in the CO2 complex is much broader than those with H2 and N2 ligands. This may be related to the fact that CO2 is the most strongly bound of the three and its attachment required operating the trap at a much higher temperature (100 versus 10 K for CO2 and H2, respectively). As such, the breadth could simply result from thermal excitation of soft modes that modulate the C5 HB linkage. It is also possible, however, that for more strongly bound ligands, excitation of the H-bonded NH stretch can be accompanied by a combination band structure involving the motion of the neutral molecule against the ion.51−54 This selective activation of tag motion upon excitation of ionic H-bonded OH stretches has been reported for anionic systems (F−·H2O),55 and traced to the charge-transfer character of the strong vibrational transition moment. The fact that the increased broadening is quite large in the CO2 case raises the question of the extent to which the widths of the H2 and N2 adducts are also due to the tag. The observation that the width does not increase upon addition of the second H2 molecule suggests that the breadth is largely an intrinsic property of the isolated ion. As such, it is clearly desirable to find systems that can be studied without a tag, presumably involving a remote UV chromophore, to definitively sort out the contribution of the tag to the breadth. At a minimum, the results presented here establish that the breadth is retained when the ions are cryogenically cooled, and this property must be taken into account when analyzing the structural implications of spectra obtained by tagging. On a qualitative level, however, the key observation of the solvation study is that the entire diffuse band evolves as would be expected for the NH stretch involved in the C5 HB linkage, raising the issue of the origin of the 1350 cm−1 (* in Figure 1a) previously suggested for this assignment. Unfortunately, several broad features close to that location are also observed in the deuterated isotopologue in the region usually associated with CH2 bends and wags. This indicates that anharmonic effects are also in play in this region of the spectrum, complicating any assignment scheme based on harmonic predictions. We next turn to the origin of the selective broadening of the C5 HB oscillator strength. In an earlier study of the sixmembered ring systems based on the protonated 1,8disubstituted naphthalene systems,56 we showed that the complicated (but discrete) band structure in regions of the shared proton (or deuteron) stretching vibrations can be rationalized in the context of anharmonic couplings introduced by cubic terms in the potential energy expansion. In this model, nominally forbidden overtones and combination bands involving shared proton bending fundamentals interact with the zero-order stretching fundamental (i.e., the “bright state”) and borrow oscillator strength from the mixing. The key feature of those systems that yield a large number of extra bands rather

explored in our earlier report on the linear IHB systems (R1R2O···H+···OR3R4), where the bridging proton bands were followed as a function of alkyl groups.18,49 Such an effect would, for example, account for the higher energy of the C5 HB feature in SarGlyH+, as discussed above. In addition, an interesting aspect of the analogous H2O···H+···OH2 or “Zundel” scaffold is that the terminal OH groups are the preferred docking sites of weak Lewis base adducts (e.g., H2O, H2, rare gas). Solvation at these sites was also found to increase the effective basicity of the water molecule accepting the base, again resulting in a blueshift of the IHB band that reflects the degree of asymmetrical solvation (i.e., more adducts on one water than the other in the Zundel core ion). In GlyGlyH+, the nonbonded NH groups on the protonated amine involved in the C5 HB play a similar role, where adduct attachment has been shown to similarly preferentially occur at these sites.47 To explore the solvent dependence of the putative C5 HB signature, Figure 3 displays the spectra of GlyGlyH+·(R)n,

Figure 3. Vibrational predissociation spectra of GlyGlyH+ tagged with (a) H2, (b) 2H2, (c) N2, and (d) CO2. All spectra were collected by monitoring the photoevaporation of all tag molecules, and the trap temperature was held at 10, 25, and 100 K for the H2, N2, and CO2 tagging experiments, respectively. For GlyGlyH+·H2, the 1600−1900 cm−1 range was collected for the singly D2-tagged complex. All spectra were taken in a laser power regime that resulted in saturation of the intense NH and OH stretching peaks to enhance the signal-to-noise of the weaker C5 HB features. Note that several additional transitions are observed in the CO2 tagged spectrum, two of which are derived from the vibrations of the CO2 molecule, while the (*)-transition is possibly derived from another isomer of GlyGlyH+.

where R = H2, N2, and CO2 with PA values of 422.3, 493.8, and 540.5 kJ/mol, respectively.50 Although the CO, spectator OH, and embedded amide NH stretches are insensitive to solvation, the C5 HB transition (red in Figure 3) shifts higher in energy (by ∼200 cm−1) with increasing PA of the solvent base. Note that as this occurs, the stretching fundamental of the NH group to which the adduct is attached (blue in Figure 3) falls lower in energy by about 100 cm−1 and broadens, as expected for complexation with increasingly basic adducts. This type of anticooperativity has been observed in a previous study on the protonated dipeptide derivative SarSar,48 where two types of isomers containing different H-bonding 3453



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state. The affect of these couplings on other regions of the spectrum are further described in the Supporting Information (section III). The diagonal energy was estimated using secondorder perturbation theory,56 which places the shared proton and shared deuteron fundamentals at 2424 and 1988 cm−1 (Figure 4, red arrows), respectively, significantly below the harmonic fundamentals at 2801 and 2046 cm−1 (B3LYP/6311++G(d,p), scaled by 0.987), respectively. Like the situation encountered in the six-membered ring systems, the shifts and breadths of the C5 HB features are thus anticipated to be strongly mixed with background states. In the peptide case, however, although the deuterium isotopic variation displays substructure that reflects some of the discrete nature of this mixing, the C5 HB feature in the light isotope (Figure 4a) is broader than anticipated at this level of theory such that the band appears more Lorentzian in shape. This additional broadening may be a consequence of higher-order terms in the potential energy expansion or activation of very soft modes associated with excitation of the tag molecule and in any case warrants further theoretical study. In summary, the vibrational predissociation spectra of several small peptides indicates that while most bands are reasonably approximated by harmonic frequency calculations, the NH stretching fundamental of the positively charged group involved in the cyclic intramolecular C5 H-bond to the proximal CO is uniquely broadened over hundreds of wavenumbers. The entire envelope is observed to behave as expected, however, in response to chemical substitutions and adduct association at the N-terminus. The implication of these findings is that the inversion of peptide ion structure from the spectra is likely intrinsically complicated by the fact the key linkages driving the structure are not readily apparent in their spectra and cannot even qualitatively be treated at the harmonic level. It would be very useful as a future direction to identify the topological features of the potential energy surface that signal when a single oscillator is subject to very strong IVR broadening while higherenergy oscillators are relatively isolated.

than just the in-and out-of-plane bend overtones is that the bending displacements of the bridging proton are distributed among many normal modes whose energies lie close to that of typical bending fundamentals. This is in contrast to the situation in the linear IHB systems where the bending displacement tends to be more localized to a few modes perpendicular to the linkage. As we have previously noted,56 the complex band patterns in the six-membered rings could only be recovered when the energy of the zero-order bright state is empirically lowered to account for mechanical anharmonicity of the one-dimensional oscillator, as noted earlier in anharmonic treatments of the OH stretching region of the organic acid dimers.57 It is important to note the implicit assumption that the off-diagonal matrix elements remain constant as one of the diagonal elements is adjusted. We have justified this approximation56 based on the observation that the interaction matrix elements for the OH bend−stretch Fermi resonances in Cl−·H2O·(CCl4)n were observed to be relatively constant.58 Furthermore, in the spectrum of the formate ion, similar behavior has been observed in the case of the CH bend−stretch Fermi resonance where microsolvation sequentially blue-shifted the CH stretching fundamental by hundreds of wavenumbers.59 Figure 4 presents the predicted GlyGlyH+ and GlyGlyD+ spectra in the region of the broad C5 H(D)B transition after inclusion of the cubic couplings to overtones and combination bands with zero-order energies falling within 1000 cm−1 of the energy corresponding to the “bright” H-bonded NH stretch

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EXPERIMENTAL DETAILS Vibrational spectra of mass-selected gas-phase ions were acquired by predissociation of weakly bound mass “tags” (e.g., H2 or N2) using the Yale tandem time-of-flight photofragmentation mass spectrometer described previously.60 Protonated peptides and derivatives were generated using a custom electrospray ionization source, where the detailed experimental parameters involving ion generation and H2 tagging have been discussed at length in previous reports.47,61 Two salient points of this study are included in this section. The first pertains to the tagging of ionic complexes with N2 and CO2, and the second is the method used to deuterate complexes. When N2 and CO2 were used as tags, the trap temperature was held at 25 and 100 K, respectively, values that were empirically optimized to maximize both the tagging efficiency as well as ensure the stable operation of the ion trap over the period of many hours. In experiments utilizing N2 as a tag, the buffer gas consisted of trace N2 in He passed over a gas trap cooled to liquid nitrogen temperatures. When CO2 tagging was employed, the buffer gas consisted of ∼10% CO2 in a balance of He that was directly pulsed into the ion trap without any precooling of the gas. Deuteration of the ions was efficiently carried out by introducing D2O vapor through a bleed valve into the second differentially pumped stage, nominally held at ∼0.20 mbar, resulting in a pressure increase

Figure 4. The experimental predissociation spectra of (a) GlyGlyH+ and (c) GlyGlyD+ in the shared proton (deuteron) regions are compared to anharmonic frequency spectra. The calculated spectra were simulated by including third-order terms in the potential energy surface and are presented in (b) and (d) for the protonated and deuterated isotopologues, respectively. The red arrows indicate the energies of the zero-order NH(D) stretching bright states in the interaction matrix that were obtained using second-order perturbation theory (see the text and Supporting Information). 3454



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of ∼0.13 mbar in this region. Vibrational spectra were collected by intersecting the mass-selected ion packet with tunable infrared radiation provided by a pulsed OPO/OPA laser system (LaserVision). To ensure that spectra were collected in a linear regime, the output power of the OPA was adjusted such that the photoinduced fragmentation was 10−15% of the initial ion intensity. The raw photofragment signal was then normalized by the output power of the laser to account for large fluctuations over the scanned range. Minimum-energy structures, harmonic frequency spectra, and cubic couplings were calculated using Gaussian 09,62 and details regarding the simulation of the anharmonic frequency spectra can be found in the Supporting Information.

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(4) Headrick, J. M.; Diken, E. G.; Walters, R. S.; Hammer, N. I.; Christie, R. A.; Cui, J.; Myshakin, E. M.; Duncan, M. A.; Johnson, M. A.; Jordan, K. D. Spectral Signatures of Hydrated Proton Vibrations in Water Clusters. Science 2005, 308, 1765−1769. (5) Cheng, T. C.; Bandyopadhyay, B.; Mosley, J. D.; Duncan, M. A. IR Spectroscopy of Protonation in Benzene−Water Nanoclusters: Hydronium, Zundel, and Eigen at a Hydrophobic Interface. J. Am. Chem. Soc. 2012, 134, 13046−13055. (6) Nagornova, N. S.; Rizzo, T. R.; Boyarkin, O. V. Interplay of Intraand Intermolecular H-Bonding in a Progressively Solvated Macrocyclic Peptide. Science 2012, 336, 320−323. (7) Jiang, J.-C.; Wang, Y.-S.; Chang, H.-C.; Lin, S. H.; Lee, Y. T.; Niedner-Schatteburg, G.; Chang, H.-C. Infrared Spectra of H+· (H2O)5−8 Clusters: Evidence for Symmetric Proton Hydration. J. Am. Chem. Soc. 2000, 122, 1398−1410. (8) Dopfer, O.; Olkhov, R. V.; Roth, D.; Maier, J. P. Intermolecular Interaction in Proton-Bound Dimers. Infrared Photodissociation Spectra of Rg-HOCO+ (Rg=He, Ne, Ar) Complexes. Chem. Phys. Lett. 1998, 296, 585−591. (9) Solca, N.; Dopfer, O. Hydrogen-Bonded Networks in Ethanol Proton Wires: IR Spectra of (EtOH)QH+-Ln Clusters (L = Ar/N2, Q ≤ 4, n ≤ 5). J. Phys. Chem. A 2005, 109, 6174−6186. (10) Hamashima, T.; Li, Y. C.; Wu, M. C. H.; Mizuse, K.; Kobayashi, T.; Fujii, A.; Kuo, J. L. Folding of the Hydrogen Bond Network of H+(CH3OH)7 with Rare Gas Tagging. J. Phys. Chem. A 2013, 117, 101−107. (11) Mizuse, K.; Mikami, N.; Fujii, A. Infrared Spectra and Hydrogen-Bonded Network Structures of Large Protonated Water Clusters H+(H2O)n (n = 20−200). Angew. Chem., Int. Ed. 2010, 49, 10119−10122. (12) Asmis, K. R.; Pivonka, N. L.; Santambrogio, G.; Brümmer, M.; Kaposta, C.; Neumark, D. M.; Wöste, L. Gas-Phase Infrared Spectrum of the Protonated Water Dimer. Science 2003, 299, 1375−1377. (13) Heine, N.; Fagiani, M. R.; Rossi, M.; Wende, T.; Berden, G.; Blum, V.; Asmis, K. R. Isomer-Selective Detection of Hydrogen-Bond Vibrations in the Protonated Water Hexamer. J. Am. Chem. Soc. 2013, 135, 8266−8273. (14) Moore, D. T.; Oomens, J.; van der Meer, L.; von Helden, G.; Meijer, G.; Valle, J.; Marshall, A. G.; Eyler, J. R. Probing the Vibrations of Shared, OH+O-Bound Protons in the Gas Phase. ChemPhysChem 2004, 5, 740−743. (15) Fridgen, T. D.; MacAleese, L.; Maitre, P.; McMahon, T. B.; Boissel, P.; Lemaire, J. Infrared Spectra of Homogeneous and Heterogeneous Proton-Bound Dimers in the Gas Phase. Phys. Chem. Chem. Phys. 2005, 7, 2747−2755. (16) Ung, H. U.; Moehlig, A. R.; Khodagholian, S.; Berden, G.; Oomens, J.; Morton, T. H. Proton-Bridge Motions in Amine Conjugate Acid Ions Having Intramolecular Hydrogen Bonds to Hydroxyl and Amine Groups. J. Phys. Chem. A 2013, 117, 1360−1369. (17) O’Brien, J. T.; Prell, J. S.; Steill, J. D.; Oomens, J.; Williams, E. R. Changes in Binding Motif of Protonated Heterodimers Containing Valine and Amines Investigated Using IRMPD Spectroscopy between 800 and 3700 cm−1 and Theory. J. Am. Chem. Soc. 2009, 131, 3905− 3912. (18) Roscioli, J. R.; McCunn, L. R.; Johnson, M. A. Quantum Structure of the Intermolecular Proton Bond. Science 2007, 316, 249− 254. (19) Stearns, J. A.; Guidi, M.; Boyarkin, O. V.; Rizzo, T. R. Conformation-Specific Infrared and Ultraviolet Spectroscopy of Tyrosine-Based Protonated Dipeptides. J. Chem. Phys. 2007, 127, 154322. (20) Vaden, T. D.; de Boer, T. S. J. A.; Simons, J. P.; Snoek, L. C.; Suhai, S.; Paizs, B. Vibrational Spectroscopy and Conformational Structure of Protonated Polyalanine Peptides Isolated in the Gas Phase. J. Phys. Chem. A 2008, 112, 4608−4616. (21) James, W. H.; Buchanan, E. G.; Muller, C. W.; Dean, J. C.; Kosenkov, D.; Slipchenko, L. V.; Guo, L.; Reidenbach, A. G.; Gellman, S. H.; Zwier, T. S. Evolution of Amide Stacking in Larger γ-Peptides:

ASSOCIATED CONTENT

S Supporting Information *

Details regarding the calculation of the anharmonic spectra, anharmonic spectra of GlyGlyH+ and GlyGlyD+ over a broader spectral range, and a table providing the assignments of the doorway states that contribute most to particular bands in the anharmonic spectra of GlyGlyH+ and GlyGlyD+. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address §

C.M.L.: Department of Chemistry, University of Georgia, Athens, GA 30602. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.B.M. would like to thank the National Science Foundation for funding under CHE-1213347. M.A.J. thanks the National Science Foundation under CHE-1213634 and the Air Force Office of Scientific Research (FA-9550-09-1-0139) for development of the cryogenic ion spectrometer essential for this work. C.J.J. acknowledges support from the NSF American Competitiveness in Chemistry Fellowship (Grant CHE1137404). M.V.S. would like to thank the Bayer School of Natural and Environmental Science and Duquesne University for start-up funds. This work was supported in part by the facilities and staff of the Yale University Faculty of Arts and Sciences High Performance Computing Center and by the National Science Foundation under CNS-08-21132 that partially funded acquisition of the facilities. We also acknowledge Arron Wolk and Joseph Fournier for their help in data acquisition.

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