Effect of phenol and acidic side chains on the protonation sites of b2 ions confirmed by IRMPD spectroscopy

International Journal of Mass Spectrometry 330–332 (2012) 144–151

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Effect of phenol and acidic side chains on the protonation sites of b2 ions confirmed by IRMPD spectroscopy Da Wang, Kerim Gulyuz, Corey N. Stedwell, Long Yu, Nicolas C. Polfer ∗ Department of Chemistry, University of Florida, Box 117200, Gainesville, FL 32611, USA

a r t i c l e

i n f o

Article history: Received 4 May 2012 Received in revised form 13 August 2012 Accepted 1 October 2012 Available online 11 October 2012 Keywords: IRMPD spectroscopy Oxazolone Diketopiperazine Peptide fragmentation Collision-induced dissociation OPO

a b s t r a c t Infrared multiple photon dissociation (IRMPD) spectroscopy was used to identify the chemical structures of b2 fragments formed by collision induced dissociation (CID) from the protonated tripeptides YGG, GYG, EGG and GEG. The IR spectra in the hydrogen stretching region (2670–3800 cm−1 ) were obtained by photodissociating the mass-selected b2 ions with a tunable benchtop optical parametric oscillator (OPO). On weaker bands, the photodissociation yield was boosted by simultaneously irradiating with a fixed wavelength CO2 laser. A comparison of the OH and NH stretching vibrations of these b2 ions to cyclo(GY)H+ and cyclo(GE)H+ reference compounds confirms that “head-to-tail” diketopiperazine structures are not formed. While the comparison of the experimental results to computed linear absorption spectra is more challenging in these systems, the trends in the NH stretching modes still allow a distinction between oxazolone structures protonated at the N-terminal N or the oxazolone ring N. It is shown that proton attachment at the oxazolone ring N is often preferred, but that an aromatic or acidic side chain can stabilize protonation at the N-terminus. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In collision-induced dissociation (CID) of protonated peptides, b2 ions are typically the smallest b fragments that are formed. The formation of b ions involves cleavage of a backbone amide bond, which is driven by nucleophilic attacks. Mechanistically, two chemical pathways need to be considered: a nucleophilic attack onto the backbone carbonyl C can occur either (1) from a carbonyl O, resulting in a five-membered oxazolone structure [1], or (2) can occur from the N-terminus, giving rise to a six-membered “head-to-tail” diketopiperazine structure [2]. To confirm the fragment structures, including the protonation site(s), infrared multiple photon dissociation (IRMPD) spectroscopy [3–5] has been applied as a useful method. Chemical moieties can be confirmed by virtue of diagnostic vibrational bands. Oxazolone structures have been identified based on the presence of an oxazolone CO stretch in the higher-frequency region of the mid-IR spectrum (1780–1950 cm−1 ) [6–14], while “head-to-tail” b fragments [15] have been confirmed for other systems based on a proton bending mode at ∼1440 cm−1 [7,11,16–17]. For most b2 ions, the spectroscopic evidence has exclusively confirmed oxazolone structures. The exception to this rule has been b fragments that include basic residues, such as histidine [14,18,19]

∗ Corresponding author. E-mail address: [email protected]fl.edu (N.C. Polfer). 1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijms.2012.10.001

and arginine [13]. Other experimental methods in mass spectrometry to elucidate the underlying mechanism in CID include ion mobility mass spectrometry [20], gas-phase hydrogen/deuterium exchange [11,12,21,22] and guided ion beam mass spectrometry [23]. Most IRMPD spectroscopic studies have been carried out at the free electron laser (FEL) facilities FELIX [24] in Netherlands or CLIO in France [25]. FELs are suitable to carry out IRMPD spectroscopy measurements, due to their intense (>MW) and widely tunable output (50–2000 cm−1 ). In addition, the pulse structure of an FEL, involving 1000s of micropulses at a high repetition rate, is ideal for promoting absorption of multiple photons [26]. The development of benchtop optical parametric oscillator/amplifier (OPO/A) light sources has provided the coverage of the hydrogen stretching region (2200–4000 cm−1 ) in infrared photodissociaton spectroscopy of gas-phase complexes [27–33]. In recent years, these light sources have been applied to assist IRMPD spectroscopy experiments on metal- and/or water-tagged weaklybound complexes [34–42]. Using a custom-built mass spectrometer, where photodissociation is carried out in a reduced-pressure (∼10−5 mbar) quadrupole ion trap, we have shown that covalently bound molecules can be routinely photodissociated [43,44]. A previous study on the b2 fragment from protonated Gly-Gly-Gly using this set-up confirmed that exclusively oxazolone structures were formed [45]. Other studies, employing an OPO light source in combination with a fixed

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wavelength CO2 laser in an ion cyclotron resonance trap [46], or in combination with a UV laser in a cold trap [47], underscore the usefulness of this region of the spectrum for structural interrogation of CID products. In this work, we implement the custom-built mass spectrometer to measure the IRMPD spectra of b2 fragment from protonated GYG, YGG, GEG and EGG to distinguish between oxazolone and diketopiperazine structures, as well as sites of proton attachment. 2. Experimental and calculations Almost all experiments were carried out using a tunable, continuous-wave (cw) optical parametric oscillator/amplifier (OPO/A) light source (LINOS Photonics OS4000) coupled to a custom-built mass spectrometer, as described in our previous studies [44,45]. This set-up includes a commercial electrospray ionization (ESI) source (Analytica, Branford, CT) to form protonated peptides, a quadrupole mass filter (Ardara Technologies, Monroeville, PA) to select the mass of the precursor ion of interest, a reduced-pressure (∼10−5 mbar) quadrupole ion trap (QIT) to allow irradiation by focused IR beams for extended periods of time (i.e., second), and a time-of-flight (ToF) drift tube (Jordan TOF Products, Grass Valley, CA) to obtain the mass spectrum of the remaining precursor and photodissociation products. The peptides GYG, YGG, GEG and EGG were made using Fmoc solid-phase synthesis. These peptides were protonated and ionized by ESI in 49:49:2 water/methanol/trifluoroacetic acid solutions at 100 ␮M. The b2 fragments were obtained through CID via “nozzle-skimmer” fragmentation in the ion source by controlling the voltage drop between the capillary exit and the skimmer. These b2 fragments were trapped in the QIT and subjected to irradiation from the tunable idler beams from the benchtop OPO light source. All the spectra were recorded using 2 s irradiation, except for the spectrum of b2 -GEG, where 3.5 s irradiation was employed. The abundances of precursor vs. photofragment ions were measured by integrating the ToF mass spectral peaks with in-house LabViewTM software. Each IRMPD spectrum was  obtained by plotting the IRMPD yield, defined as yield = −ln[1 − (photofragments)/ (photofragments + precursor)], versus OPO wavenumber in cm−1 . In some cases, the IRMPD yield was boosted by simultaneously irradiating with a fixed wavelength CO2 laser at 10.6 ␮m (=943 cm−1 ) shuttered at 10 Hz, alternately irradiating for 20 ms, followed by no irradiation for 80 ms. In principle, non-resonant excitation with the CO2 laser could lead to isomerization issues, in that the original conformer population in the ion trap is heated by the CO2 laser, and hence disturbed. The shuttered pulse structure of the CO2 laser, providing for cool-down periods (of 80 ms), is effected to minimize this effect. The potential heating effects of the CO2 laser on the present results will also be discussed in the Results and Discussion section for the example of the b2 -YGG results. The peptides cyclo(GY) and cyclo(GE) (Bachem, Torrance, CA) were protonated by ESI, and then their IRMPD spectra were measured using the same method described above, without the need to generate a fragment ion. A control IRMPD experiment on protonated cyclo(GY) was carried out at the FOM institute for Plasma Physics ‘Rijnhuizen’, using a pulsed OPO/A (Laser Vision, Bellevue, WA) tethered to their laboratory-built Fourier transform ion cyclotron resonance (FTICR) mass spectrometer [33,48]. In those measurements, the protonated cyclo(GY) ions were generated by ESI, trapped in the ICR cell, where they were irradiated with 90 pulses. For each b2 fragment from the protonated peptides described above, three chemical structures need to be considered computationally: oxazolone protonated on the oxazolone ring N (oxazolone ox-prot), oxazolone protonated on the N-terminus (oxazolone Nprot), and the diketopiperazine structure protonated on a carbonyl

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Table 1 Electronic and ZPE-corrected energies at the B3LYP/6-31g** level of theory, and ZPE-corrected energies at the B3LYP/6-311g** level of theory, for the lowest-energy conformers shown in Figs. 2–8. Structures

b2 -GYG (A) b2 -GYG (B) b2 -YGG (A) b2 -YGG (B) b2 -GEG (A) b2 -GEG (B) b2 -GEG (C) b2 -EGG (A) b2 -EGG (B) cyclo(GY)H+ (A) cyclo(GY)H+ (B) cyclo(GE)H+ (A) cyclo(GE)H+ (B) cyclo(GE)H+ (C)

B3LYP/6-31g**

B3LYP/6-311g**

Etotal /H

Etotal + ZPE/H

Etotal + ZPE/H

−761.97890 −761.98983 −761.98607 −761.98537 −683.59701 −683.60237 −683.59623 −683.60304 −683.59478 −762.00142 −761.99589 −683.61879 −683.61282 −683.77692

−761.74217 −761.75533 −761.74996 −761.75048 −683.40135 −683.40946 −683.40290 −683.40839 −683.40308 −761.76551 −761.76024 −683.42439 −683.41859 −683.58287

−761.91082 −761.92431 −761.91862 −761.91930 −683.56419 −683.57255 −683.56599 −683.57089 −683.56646 −761.93406 −761.92899 −683.58639 −683.58121 −683.55700

O (diketopiperazine O-prot). The geometry optimizations and frequency calculations of all the structures in this study were carried out using the B3LYP/6-31g** level of theory and the higher 6-311g** basis set. All energies in the figures are reported as zero-point energy (ZPE)-corrected energies, using the frequency calculations at the B3LYP/6-31g** level. The harmonic frequency spectra at the B3LYP/6-31g** level of theory were scaled by 0.961, as recommended by Pearce et al. [49], and the stick spectra were convoluted by a Gaussian function (full-width at half-maximum = 20 cm−1 ) to allow easier comparisons with the experimental spectra. The computations using the extended 6-311g** basis set are shown in the Supplementary Materials using the scaling factor of 0.9663 as suggested by Tas¸al et al. [50]. The calculations are summarized in Table 1. 3. Results and discussion 3.1. cyclo(GY)H+ , b2 -YGG and b2 -GYG 3.1.1. IRMPD spectroscopy results The IRMPD spectra of cyclo(GY)H+ , b2 -YGG and b2 -GYG are compared in Fig. 1. The results for cyclo(GY)H+ serve as a control experiment, as it is structurally identical to the diketopiperazine structure. All three spectra exhibit a strong band around 3640 cm−1 , which corresponds to the OH stretch of the phenol group. However, the remainder of the spectra for b2 -YGG and b2 -GYG show no overlap with the spectral signature of the diketopiperazine structure (i.e., cyclo(GY)H+ ), thus confirming that neither b2 fragment adopts the diketopiperazine structure. In addition, the vibrational bands for b2 -YGG and b2 -GYG display different patterns. Both spectra display prominent bands centered at ∼3340 cm−1 ; however, b2 -GYG has two smaller satellite peaks at higher frequency, whereas b2 YGG has a broader feature centered at 3150 cm−1 . The diagnostic modes are indicated by color-coding, and will be discussed hereafter, based on a comparison to theory. 3.2. Comparison to theory A comparison of the experimental IRMPD spectrum for b2 -GYG to calculated spectra of oxazolone ox-prot and oxazolone N-prot is shown in Fig. 2. The thermochemistry suggests that protonation on the oxazolone ring nitrogen is favored (by a substantial 34.5 kJ mol−1 ). Experimentally, the vibrational signature closely resembles the one previously recorded for b2 -GGG by Wang et al. [45], which had been assigned to the oxazolone ox-prot structure. The similarities in these characteristic experimental patterns, in

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Fig. 2. IRMPD spectrum of b2 -GYG compared with the lowest-energy conformers for (A) oxazolone N-prot and (B) oxazolone ox-prot. The inset shows the previously recorded IRMPD spectrum for b2 -GGG, assigned to the oxazolone ox-prot structure. Zero-point energy (ZPE)-corrected energies at the B3LYP/6-31g** level are shown. Fig. 1. IRMPD spectra of (A) protonated cyclo(GY), (B) b2 from protonated YGG, and (C) b2 from protonated GYG. The most prevalent structure is shown, along with color-coding of the diagnostic modes. The insets show CO2 -assisted photodissociation to enhance the weaker bands, or to confirm the absence of a band at a particular position. The corresponding photodissociation percentage is shown on the right-hand-side.

terms of the protonated oxazolone ring NH stretch (measured at 3340 cm−1 ), as well as the symmetric and antisymmetric stretching modes for the amine NH2 (measured at 3400 and 3480 cm−1 ), are also borne out by the computed IR spectra for oxazolone oxprot. Note that the lower-frequency CH stretches, predicted at ∼2925 and 3050 cm−1 are often too weak to be observed by IRMPD spectroscopy. On the other hand, the oxazolone N-prot structure should be validated by the presence of an intense mode associated with the NH3 + moiety, predicted here at 3120 cm−1 . A previous IRMPD spectroscopy study for protonated tryptophan had shown that NH3 + stretching modes can give rise to broad features (e.g., 2950–3200 cm−1 ) [44], due to the conformational flexibility of room-temperature ions, and the multiple-photon absorption process in IRMPD. Clearly, no lower-frequency modes are observed anywhere between 2800 and 3300 cm−1 , strongly indicating that the oxazolone N-prot structure is absent. Thus, we conclude that the oxazolone ox-prot structure is dominant in gas-phase b2 -GYG. Fig. 3 shows a comparison of the experimental spectrum for b2 YGG to the computed spectra for oxazolone ox-prot and oxazolone N-prot. While similarly to b2 -GYG, an intense band is observed at 3340 cm−1 , the characteristic symmetric and antisymmetric NH2 modes are missing. Conversely, a broader feature is observed from 3100 cm−1 to 3200 cm−1 , consistent with the presence of NH3 + modes predicted at 3137 and 3198 cm−1 for oxazolone N-prot. In

the context of the experimental results for b2 -GYG, the spectral signature for b2 -YGG thus appears to confirm the oxazolone Nprot structure, while excluding the oxazolone ox-prot structure. This also implies a different assignment of the prominent bands at 3340 cm−1 , observed in both IRMPD spectra: for b2 -GYG the 3340 cm−1 band is associated with the protonated oxazolone ring NH stretch, whereas for b2 -YGG the 3340 cm−1 band is assigned to the antisymmetric NH3 + stretch mode. A detailed assignment of the experimental to computed bands for b2 -GYG and b2 -YGG is given in Table 2. In spite of the strong spectroscopic evidence for oxazolone Nprot, the thermochemistry results are within the margin of error. In the oxazolone N-prot structure, the bond distance between the ˚ and NH3 + group and the tyrosine side chain is rather large (∼5 A), is hence expected to stabilize solvation of the charge on the Nterminus, but rather weakly. An identical b2 CID product with Table 2 Assignment of vibrational bands for b2 -GYG and b2 -YGG compared to theoretical predictions of best-matching structures in Figs. 2 and 3. Experimental band position/cm−1

Assignment

b2 -GYG 3649 3453 3395 3342 b2 -YGG 3644 3337 3100–3200

Oxazolone ox-prot Phenol OH stretch Antisymmetric NH2 stretch Symmetric NH2 stretch Oxazolone NH stretch Oxazolone N-prot Phenol OH stretch Antisymmetric NH3 + stretch NH3 + stretches

Scaled frequencies at B3LYP/6-31g**/cm−1 3662 3499 3417 3380 3667 3364 3198, 3137

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Fig. 3. IRMPD spectrum of b2 -YGG compared to the computed spectra of the lowest-energy conformers for (A) oxazolone N-prot and (B) oxazolone ox-prot. ZPE-corrected energies at the B3LYP/6-31g** level are shown.

sequence YG, generated from protonated Leu-enkephalin (YGGFL), had been recorded previously, also confirming that a majority of oxazolone N-prot structures were formed [12]. In that study, a minor presence of oxazolone ox-prot structures was also confirmed, based on a higher-frequency shoulder of the structurally diagnostic oxazolone CO stretch. Despite CO2 -assisted photodissociation (see Fig. 1B), the structurally diagnostic symmetric and antisymmetric NH2 modes for oxazolone ox-prot could not be detected for b2 -YGG. This suggests that the ions in the quadrupole ion trap (QIT) are cooler than in the corresponding FELIX spectroscopy measurements in an ion cyclotron resonance (ICR) trap, consequently leading to exclusive detection of oxazolone N-prot in the present results. In fact, the heating effect of the CO2 laser could in principle lead to a small population of oxazolone ox-prot structures. The fact that this is not observed also supports the hypothesis that CO2 -assisted photodissociation does not lead to isomerization. It should be noted that our comparative results between IRMPD experiments in a QIT and an ICR trap are analogous to a previous study by Simon et al. [51], where the IRMPD bands of ions trapped in a QIT were found to be narrower than the corresponding bands for ions photodissociated in an ICR trap.

Fig. 4. IRMPD spectra of (A) protonated cyclo(GE), (B) b2 from protonated EGG, and (C) b2 from protonated GEG. The most prevalent structure(s) is/are shown, along with color-coding of the diagnostic modes.

two amide NH stretches are located around 3400 cm−1 . The spectral signature of cyclo(GE)H+ is not reproduced for either b2 -EGG or b2 -GEG, thus excluding the diketopiperazine structure as a significant component in these b ions. Once again, the IRMPD spectra for these b fragments display considerable differences in their NH stretching vibrations, indicating differences in their protonation sites. The spectral features for b2 -EGG are clearly reminiscent of an oxazolone N-prot structure, given the absence of NH2 modes, as well as the presence of characteristic NH3 + stretch modes, in particular the isolated feature at 3340 cm−1 (antisymmetric NH3 + stretch), and the very broad band from 2800-3200 cm−1 (lowerfrequency NH3 + stretches). The picture is less clear-cut for b2 -GEG. On the one hand, the characteristic symmetric and antisymmetric NH2 stretches at 3400 and 3460 cm−1 are indicative for the oxazolone ox-prot structure. Conversely, the broad tail from 3100 to 3250 cm−1 is more ambiguous to interpret at first sight. 3.4. Comparison to theory

3.3. cyclo(GE)H+ , b2 -EGG and b2 -GEG 3.3.1. IRMPD spectroscopy results The IRMPD spectra of cyclo(GE)H+ , b2 -EGG and b2 -GEG are shown in Fig. 4. All three experimental IRMPD spectra have strong peaks located in the same region around 3550 cm−1 , corresponding to the OH stretch of the acetic acid group. The OH band for cyclo(GE)H+ contains an incompletely resolved shoulder on its red side, which is assigned to the protonated carbonyl OH stretch. The

A comparison between experiment and computations for possible oxazolone structures for b2 -GEG is shown in Fig. 5. The observed NH2 symmetric and antisymmetric stretches at 3400 cm−1 and 3460 cm−1 give compelling evidence for the oxazolone ox-prot structure, yet it is not immediately apparent how the oxazolone ring NH stretch would rationalize the very broad feature from 3100 to 3370 cm−1 . Spectroscopic evidence for b2 -GGG and b2 -GYG had shown that the oxazolone ring NH stretch is typically centered at

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Fig. 6. IRMPD spectrum of b2 -EGG compared with the lowest-energy conformers for (A) oxazolone N-prot and (B) oxazolone ox-prot. ZPE-corrected energies at the B3LYP/6-31g** level are shown.

Fig. 5. IRMPD spectrum of b2 -GEG compared with the lowest-energy conformers for (A) oxazolone N-prot and (B) oxazolone ox-prot. ZPE-corrected energies at the B3LYP/6-31g** level are shown.

3340 cm−1 with a full-width at half-maximum (FWHM) of 35 cm−1 . Nonetheless, the glutamic acid carboxylic acid side chain offers an attractive interaction partner for the oxazolone NH group, and is thus likely to affect the oxazolone NH stretch in different conformations. In support of this hypothesis, the NH stretch for the tightly chelated geometry (Fig. 5B) predicted at ∼3240 cm−1 , is blueshifted to 3340 cm−1 in the more loosely chelated structure (Fig. 5C). A mixture of different oxazolone ox-prot conformers could thus account for the broad band between 3100 and 3370 cm−1 . It should be noted though that energetically, the second-lowest energy conformer (+17.2 kJ mol−1 ) appears to be too high to be populated, and hence other low-energy conformers may have to be considered. On the other hand, the oxazolone N-prot structure is calculated to be even higher in energy (21.3 kJ mol−1 ), and its match with the experimental spectrum is poor. In summary, the data seems to be most in concordance with the oxazolone ox-prot structure. In Fig. 6, the experimental spectrum for b2 -EGG is compared to computations for oxazolone N-prot and oxazolone ox-prot structures. Protonation at the N-terminus (i.e., oxazolone N-prot) is energetically favored (by 14 kJ mol−1 ) over protonation at the oxazolone ring nitrogen (i.e., oxazolone ox-prot). Similarly to b2 YGG, where the side-chain ␲-cloud stabilizes protonation at the N-terminus, here the side-chain carbonyl O exhibits a favorable interaction with the NH3 + group. The absence of NH2 modes at

3400 and 3460 cm−1 , which were previously observed in the oxazolone ox-prot structures for b2 -GGG and b2 -GYG but are not seen here, is consistent with an absence of oxazolone ox-prot structures. The spectroscopic evidence lends some support to the presence of oxazolone N-prot structures, based on diagnostic NH3 + stretch bands. The computations predict 3 NH3 + stretch bands at 2680 cm−1 , 3240 cm−1 and 3360 cm−1 . Of these, the antisymmetric NH3 + band, observed at 3340 cm−1 , is reasonably well matched and isolated. On the other hand, the lower-frequency NH3 + bands are not well matched, and are often observed as broad, unresolved features in IRMPD spectra, such as observed for protonated tryptophan [44]. Similarly, DFT-based Born-Oppenheimer molecular dynamics simulations of the peptide Ala7 H+ [52] showed that the broad experimental features between 2800 and 3400 cm−1 could be explained by anharmonicities in the N H+ vibrations, related to hydrogen bonding interactions of the NH3 + group with backbone carbonyls. While the low-frequency NH3 + modes are hence not well reproduced by harmonic frequency calculations, past experience leads us to tentatively conclude that the vibrational features for b2 -EGG are in agreement with the presence of oxazolone N-prot structures. A detailed assignment of the experimental to computed bands for b2 -GEG and b2 -EGG is given in Table 3. 3.5. Limitations of theory Based on the comparisons of experimental and computational results in Figs. 5 and 6, it appears that the match between experiment and theory is rather tentative. This demonstrates some of the challenges in directly comparing computed linear absorption spectra of 0 K conformers to IRMPD spectra of room-temperature

D. Wang et al. / International Journal of Mass Spectrometry 330–332 (2012) 144–151 Table 3 Assignment of vibrational bands for b2 -GEG and b2 -EGG compared to theoretical predictions of best-matching structures in Figs. 5 and 6. Experimental band position/cm−1

Assignment

b2 -GEG 3566 3462 3399 3100–3360 b2 -EGG 3558 3339 2800-3150

Oxazolone ox-prot Phenol OH stretch Antisymmetric NH2 stretch Symmetric NH2 stretch Oxazolone NH stretch Oxazolone N-prot Phenol OH stretch Antisymmetric NH3 + stretch NH3 + stretches

Scaled frequencies at B3LYP/6-31g**/cm−1 3587 3509 3424 3239 3576 3364 3235, 2675

ions. This is particularly relevant for hydrogen stretching vibrations, involving the site of proton attachment, which can be shared between different parts of molecule. Previous IRMPD measurements had already shown that the low-frequency NH3 + modes are impacted by the chemical environment that this moiety interacts with [44,52]. This can in principle complicate spectral analysis, even if trends in the vibrational features between related molecular systems are often distinct enough to allow an interpretation, based on chemical intuition.

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frequency is red-shifted considerably to ∼3130 cm−1 . No experimental band is observed at this position, nor anywhere else in the range from 2680 to 3300 cm−1 in the cw OPO measurements. Conversely, in the pulsed OPO IRMPD measurements (conducted at the FOM institute ‘Rijnhuizen’), a broad band is confirmed at this position. Note that the amide NH bands are observed in both experiments between 3350 and 3450 cm−1 . The intense phenol OH stretch (observed at 3640 cm−1 ) is clearly discernible, as is a much weaker band at 3600 cm−1 . The failure to observe any OH+ stretch at 3120 cm−1 (i.e., consistent with structure A) in the cw OPO measurements is most likely ascribed to the fact that slow (i.e., ␮s–ms) absorption of multiple photons at the resonant OH+ stretch likely breaks the interaction of the proton with the phenol side chain, thus considerably blueshifting this mode, and hence preventing subsequent absorption of multiple photons. In contrast, in the pulsed OPO experiments, the timescale of the laser pulse (and thus photon absorbance) is on the order of nanoseconds, and hence faster than the time required to break this hydrogen bond. In other words, the frequency of the OH+ stretch does not blue-shift during the absorption process. This leaves the assignment of the weaker band at 3600 cm−1 , which we tentatively assign to a free OH+ stretching mode, as

3.6. Protonation of cyclic dipeptides For cyclo(GY)H+ two sites of proton attachment have to be considered, namely the glycine and tyrosine residue carbonyls. Structure A in Fig. 7, the lowest-energy conformer, involves a sharing of the proton between the carbonyl O and phenol side chain ␲-cloud. As a result of this interaction, the predicted OH+ stretch

Fig. 7. IRMPD spectrum of cyclo(GY)H+ compared with the lowest-energy conformers for diketopiperazine O-prot structures protonated on (A) tyrosine CO and (B) glycine CO. ZPE-corrected energies at the B3LYP/6-31g** level are shown. Insert shows IRMPD spectrum for cyclo(GY)H+ recorded with a pulsed OPO.

Fig. 8. IRMPD spectrum of cyclo(GE)H+ compared with the lowest-energy conformers for diketopiperazine O-prot structures, (A) protonated on glutamic acid CO, (B) protonated on glycine CO, and (C) diketopiperazine COOH-prot. ZPE-corrected energies at the B3LYP/6-31g** level are shown.

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previously observed for cyclo(GG)H+ [45]. In fact, this is the position where the OH+ stretch is predicted for structure B, where the proton is not solvated by the tyrosine side chain. Since structure B is energetically disfavored, only a small portion of the ion population would adopt such a structure, thus rationalizing the very small IRMPD yield at this frequency. In cyclo(GE)H+ , there are a priori three distinct sites of proton attachment: on either carbonyl oxygen, or the side-chain carboxylic acid. The computed linear absorption spectra for all of these structures are shown in Fig. 8. The predicted IR spectrum for the lowest-energy structure A, with proton attachment on the glutamic acid residue carbonyl, mirrors the carboxylic acid OH and amide NH bands fairly closely. The higher-energy structure B, with proton attachment on the glycine residue backbone carbonyl, yields a lesser satisfactory match. Finally, proton attachment on the glutamic acid side chain in structure C yields a poor match, and is highly disfavored energetically. Detection of the structurally diagnostic proton O H+ stretch in structure A is far redshifted. Given the complications with measuring the O H+ stretch in cyclo(GY)H+ (see above), the detection of this mode is likely beyond our current capabilities, and was hence not attempted. 4. Conclusions IRMPD spectra in the hydrogen stretching region confirm that a series of b2 ions, b2 -YGG, b2 -GYG, b2 -GEG and b2 -EGG, form oxazolone structures, as opposed to “head-to-tail” diketopiperazine structures. Moreover, characteristic patterns in the NH vibrations allow a distinction in the site of proton attachment at the Nterminus versus the oxazolone ring nitrogen, in particular the symmetric and antisymmetric NH2 stretch modes (measured as distinct peaks at 3400 and 3480 cm−1 ), and the lower-frequency NH3 + stretch modes (observed as broad features from 3100 to 3200, or even 2800–3200 cm−1 ). In spite of the strong evidence for structural interpretation in these results based on chemical intuition, the comparison to quantum-chemical calculations is at times found to be unsatisfactory, chiefly for the low-frequency NH3 + stretch modes in the b2 -GEG and b2 -EGG structures. As shown previously, these modes are highly susceptible to interactions with other moieties in the molecule, and are hence affected by the temperature of the ions, and the conformational degrees of freedom that are accessible. The results presented here underline the importance of the amino acid side chain in stabilizing the charge on the N-terminus, as opposed to protonation on the oxazolone ring nitrogen. Both the tyrosine ␲-cloud and glutamic acid carbonyl side-chain groups are found to aid solvatation of the NH3 + group, thus promoting protonation at the N-terminus in b2 -YGG and b2 -EGG. In the absence of these interactions, proton attachment on the oxazolone ring N is favored, as observed for b2 -GYG, and b2 -GEG. In terms of the overall trends of CID chemistry, a growing database of IRMPD spectra on b2 fragment ions has shown that oxazolone structures are formed most commonly, and that “head-to-tail” diketopiperazine structures are related to sequences incorporating basic amino acid residues. Acknowledgments Professor Peter Armentrout is acknowledged for his many contributions to elucidating gas-phase structures over the years, using a range of techniques, including guided ion beam mass spectrometry, ion mobility, ion spectroscopy and computational methods. This research is financially supported by the National Science Foundation under grant CHE-084545. Professor John R. Eyler is thanked for providing access to his OPO, which was funded from an In-House

Research Program (IHRP) grant from the National High Magnetic Field Laboratory (NHMFL). Dr Giel Berden and Dr Jos Oomens from the FOM institute ‘Rijnhuizen’ are acknowledged for recording the IRMPD spectrum of cyclo(GY)H+ with their pulsed OPO set-up. The authors’ collaborators from Ardara Technologies and, particularly, Randall E. Pedder and Christopher Taormina are thanked for their help in designing and setting up the custom-built mass spectrometer described here. Damon T. Allen is acknowledged for developing the LabViewTM software applied in the mass spectral analysis. Finally, the authors thank their colleagues in the mechanical and electronic workshops in the department of chemistry for all their help and, in particular, Todd Prox, Brian Smith, Joe Shalosky, and Steven Miles. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijms.2012.10.001. References [1] T. Yalcin, I.G. 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