Characterizing the Tautomers of Protonated Aniline Using Differential

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Characterizing the Tautomers of Protonated Aniline Using Differential Mobility Spectrometry and Mass Spectrometry Stephen W. C. Walker, Alison E. Mark, Brent Verbuyst, Bogdan Bogdanov, J Larry Campbell, and W. Scott Hopkins J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b10872 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Characterizing the Tautomers of Protonated Aniline using Differential Mobility Spectrometry and Mass Spectrometry

Stephen W. C. Walker,1 Alison Mark,1 Brent Verbuyst,1 Bogdan Bogdanov,2 J. Larry Campbell,1,3* and W. Scott Hopkins1*

1. Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada 2. Department of Chemistry, University of the Pacific, Stockton, CA, 95211, U.S.A. and Shimadzu Scientific Instruments, Pleasanton, CA 94566, U.S.A. 3. SCIEX, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada *Corresponding authors: [email protected]; [email protected]

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Abstract The site of protonation for gas-phase aniline has been debated for many years, with many research groups contributing experimental and computational evidence for either the aminoprotonated or the para-carbon-protonated tautomer as the gas-phase global minimum structure. Here, we employ differential mobility spectrometry (DMS) and mass spectrometry (MS) to separate and characterize the amino-protonated (N-protonated) and para-carbon-protonated (pprotonated) tautomers of aniline. We demonstrate that upon electrospray ionization (ESI), aniline is protonated predominantly at the amino position. Similar analyses are conducted on another three isotopically labeled forms of aniline to confirm our structural assignments. We observe a significant reduction of the relative population of the p-protonated tautomer when a protic ESI solvent is employed (methanol/water) compared to when an aprotic solvent (acetonitrile) is employed. We also observe conversion of the p-protonated species into the N-protonated species upon clustering with protic solvent vapor post-DMS selection – a finding supported by previous experimental data acquired using DMS-MS.

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1. Introduction Identifying the most favorable site(s) for protonation of molecules in the gas phase is a matter of both practical and fundamental importance for understanding behavior of ions.1-7 Owing to its relative simplicity and the fact that it exhibits two nearly iso-energetic sites for protonation, aniline has become an archetypical system in gas phase protonation studies.8-19 In solution, it has been well established that aniline protonates at the amino moiety due to stabilization through solvation. However, determination of the site of protonation in the gas phase has been more difficult; of the four possible protonation sites (the amino group, and the ortho, meta, and para ring positions; see Figure 1), the para-protonated (p-protonated) and amino-protonated (N-protonated) tautomers have been shown to be the two most stable species.46, 12-15, 19

Although a great deal of effort has gone into determining which of these two tautomers

is the gas phase global minimum structure, the mass spectrometry community has still to reach a consensus.

Figure 1. The four possible tautomers of protonated aniline: (Left to right) nitrogenprotonated, para-, meta-, and ortho-carbon-protonated. The nitrogen- and para-carbonprotonated tautomers have been previously observed in mass spectrometric experiments.19

Using chemical ionization methods, Wood et al. concluded that protonation at the para position is favored and suggested that the observed loss of ammonia originates from a 1,2elimination of the amino group and the hydrogen in the ortho position.19 These authors point out

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that the fragment ion spectrum is somewhat ambiguous, but go on to conduct a comprehensive isotopological study to provide further evidence for assigning the para position as the site of protonation. In another study, Pachuta et al. utilized charge stripping following CID to elucidate the protonation site and concluded that protonation most likely occurs somewhere on the carbon ring.17 However, in this work, the authors found that the N-protonated form was also present.20 Based on ion-molecule reactions, Smith et al. also concluded that the p-protonated species is the gas phase global minimum structure and that the N-protonated species is kinetically favored.12 More recently, several groups have utilized ion mobility spectrometry to study protonated aniline. Karpas et al. observed two peaks in their arrival time distribution (ATD) and concluded that both the p-protonated and N-protonated tautomers are formed following ESI.14 In this study, the N-protonated species was determined to be the most abundant tautomer in the ensemble. Conversely, Nold et al. found that under different ionization conditions (FAB vs. CI) ionization occurs at different sites, with FAB predominantly protonating aniline on the ring.16 Lalli et al. and Attygalle et al. have both used traveling wave ion mobility mass spectrometry (TWIMS) systems to investigate protonated aniline.13, 15 Both groups found evidence of two tautomers in the ATD, the fragmentation spectra of which were consistent with formation of both the pprotonated and N-protonated species. Lalli et al. found a 1:5 ratio of areas for the peaks assigned to the p-protonated and N-protonated species, respectively, and concluded that this was representative of the intrinsic population of the two tautomers because the intensity profile did not change as a function of pH. However, Attygalle et al. found that the ratio of the p- and Nprotonated peaks was dependent on the sampling-cone voltage of their instrument, thus demonstrating that post-ESI source conditions can have a major impact on observed tautomer distributions. This also raises the question as to if field-induced heating can influence relative

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tautomer populations within the mobility analyzer. For example, Shvartsburg and Smith calculated that ions in a TWIMS cell could reach temperatures as high as 5000 K.20 Merenbloom et al. studied field-induced heating in TWIMS using thermometer ions and showed that they could reach temperatures as high as 774 K.21 While this effect is expected to be small for linear IMS, it is likely that relatively high ion temperatures could also be generated by differential mobility spectrometry (DMS). Using DMS, Campbell et al. were able to separate the N-protonated and O-protonated tautomers of 4-aminobenzoic acid (4-ABA) and study the effect of the ESI solvent on the tautomer populations.22 They demonstrated that a 50:50 methanol/water solution yields a mixture of N-protonated and O-protonated tautomers, with the O-protonated species making up 92 % of the total population. Changing the ESI solvent to acetonitrile shifts the population of the Oprotonated species down to 78 % of the ensemble. Also using DMS, Anwar et al. were recently able to separate various tautomers of protonated nucleobases prior to MS characterization. This ability to separate tautomers prior to interrogation not only affords researchers the opportunity to characterize these ensemble sub-populations individually, but also enables experiments designed to monitor conditions wherein tautomers can interconvert. For example, in a separate study, Campbell et al. demonstrated that the N-protonated tautomer of 4-ABA converts to the Oprotonated tautomer upon clustering with protic solvent vapor, thus providing evidence that the O-protonated species is the gas phase global minimum structure.23 Recently, Xia et al. employed TWIMS to similarly demonstrate that kinetically trapped tautomers of protonated benzocaine could be “untrapped” by ambient humidity within the ion source.24 Here, we bring these techniques to bear on the p-protonated and N-protonated tautomers of aniline.

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2. Experimental Formic acid (~80%), aniline (C6H5-NH2), D5-aniline (C6D5-NH2), 15N-aniline (C6H5-15NH2), and

13

C6-Aniline (13C6H5-NH2) were obtained from Sigma-Aldrich (St. Louis, MO) and were

used without further purification. HPLC-grade acetonitrile (Caledon Laboratory Chemicals, Georgetown, ON) was also used without further purification. Distilled deionized water (18 MΩ) was produced in-house using a Millipore (Billerica, MA) Integral 10 water purification system. In each experiment, an analyte solution (10 ng/mL) was infused at a rate of 15 µL/min into DMS-MS instrument via an ESI source operating at 5.5 kV, with a source temperature of 300 °C, nebulizing gas pressure of 20 psi, and auxiliary gas pressure of 20 psi. The DMS cell (SelexIONTM, SCIEX, Concord, ON) system was mounted on a 5500 QTRAP® system (SCIEX), between a TurboV TM ESI source and the mass spectrometer’s sampling orifice (see Figure 2).2528

The temperature of the DMS cell was maintained at 150 °C, and nitrogen was used as the

curtain gas (3.5 L/min), throttle gas (0 or 0.7 L/min), and target gas (~3 mTorr) for the MS/MS experiments. The fundamental behavior of DMS devices is described elsewhere.27-32 For the DMS experiments conducted in this study, one of two operational modes was employed. In the first mode, the separation voltage (SV) was held at an optimum value (+3500 V, 116 Td) while the compensation voltage (CV) was scanned from −12 V to +10 V in 0.1 V increments unless otherwise indicated. This mode of operation generates an ionogram, which is used to identify specific SV/CV conditions for tautomer selection prior to HDX and tandem MS/MS collision

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induced dissociation (CID) experiments. For HDX experiments, the throttle gas was saturated with CH3OD vapor (ca. 13.3 kPa at 20 ºC). The second operational mode synchronously scans both SV and CV to monitor ion behavior across all field conditions (reported as a dispersion plot); as SV was stepped from 0 to 4000 V (in 500 V increments), CV was scanned from −12 V to +15 V at each incremental SV value.

Figure 2. Cross-sectional view of the DMS-MS source, depicting the relevant components and gas flows. Volatile chemical modifiers can be added to the curtain gas or throttle gas. Deuterated solvent vapor can be added to the throttle gas to perform HDX experiments on the DMS-separated species. Adapted from references 22 and 33.

3. Computational Experimental studies of protonated aniline were supported with parallel computational investigation. The N-protonated and p-protonated tautomers of aniline were geometry optimized at the B3LYP/6-311++G(d,p) level of theory, and partial charges were calculated using the ChelpG partition scheme.34-37 These two tautomers were then sequentially microsolvated with up

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to five molecules of water or methanol using a custom-written basin hopping (BH) search algorithm.38-41 We have used basin hopping in conjunction with experimental DMS to good success previously.23, 42-44 In this study, on each iteration of the BH routine the solvent molecules are randomly translated in each of the x, y, and z directions by a step size of -0.5 ≤ η ≤ 0.5 Å. The solvent molecules were also given a random rotation of -5o ≤ θ ≤ 5o about their body-fixed axes. In total, ca. 20,000 structures were sampled. The unique isomers identified by the BH routine were re-optimized at the B3LYP/6-311++G(d,p) level of theory including the GD3 empirical dispersion correction,45 and normal mode analyses were conducted to ensure that each geometry was associated with a local minimum on the potential energy surface and to calculate thermodynamic corrections. Calculations at the CCSD(T)/6-311++G(d,p) level of theory were conducted for clusters with up to three water molecules and two methanol molecules to improve electronic energies. To calculate the relative populations of the bare tautomers, G3MP2 calculations were performed.36, 46 For the N-, C4-, and C2-protonated tautomers, the G3MP2 free energies (hartree) were calculated at absolute temperatures from 300-800 K, in 25 K increments. Relative Gibbs’ free energies (kcal mol‒1) were calculated and the % populations of the three tautomers were determined using the Boltzmann distribution. The potential energy surface of protonated aniline as calculated at the G3MP2 level of theory is available in the supporting information that accompanies this manuscript.

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4. Results and Discussion The ionogram recorded for protonated aniline at SV = 3500 V following ESI from anhydrous acetonitrile is shown in the top panel of Figure 3. Two features are observed, indicating the presence of two distinct species with m/z 94. The peak at CV = −6.5 V is approximately thirty times larger than the feature at CV = −2.5 V, suggesting that the gas phase ensemble is dominated by one tautomeric form of protonated aniline. To confirm that each of these peaks corresponded to a different tautomeric form of protonated aniline, a variety of experiments were conducted, which were supported by high-level electronic structure calculations.

Figure 3. (Top Panel) The ionogram of natural aniline, 12C6H514NH2, recorded at SV = 3500 V. Two peaks, labeled A and B, are observed. (Middle Panel) The mass spectrum recorded following fragmentation of peak A via CID at a collision energy of 38 eV (lab frame). The dominant loss channel is 17 mass units, indicating loss of NH3. (Bottom Panel) The mass spectrum recorded following fragmentation of peak B via CID at a collision energy of 38 eV (lab frame). Several additional loss channels are observed in comparison with the spectrum recorded for peak A.

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The middle panel of Figure 3 shows the mass spectrum recorded following isolation and CID of the molecule transmitted at CV = −6.5 V (labeled peak A in Figure 3). Under the employed CID conditions, substantial fragmentation of the parent ion (m/z 94) and a single dominant fragment channel (m/z 77) was observed. The loss of 17 mass units is consistent with loss of NH3, a well-known fragmentation channel of protonated aniline and the dominant loss channel for the N-protonated tautomer. The bottom panel of Figure 3 shows the mass spectrum recorded following isolation and CID of the molecule transmitted at CV = −2.5 V (labeled peak B in Figure 3). The parent ion associated with peak B fragments to a lesser extent than does the parent ion associated with peak A at the employed CID conditions, and it produces fragments at m/z 93, 79, and 78 in addition to the m/z 77 channel. The m/z 93 and 79 fragmentation channels have been previously assigned to loss of H● and CH3●, respectively, from the p-protonated tautomer.13, 15-16

Lalli et al. proposed that loss of NH3 from the p-protonated species (i.e., production of the

m/z 77 fragment) could occur following conversion of the ring protonated tautomer into the Nprotonated species.15 It is also possible that the amino group could abstract an ortho hydrogen atom during fragmentation to produce NH3. To the best of our knowledge, the m/z 78 fragment (i.e., loss of 16 mass units) has not been previously reported. However, given that loss of H and CH3 are observed, it is possible that m/z 78 fragment arises from sequential loss of H and CH3, or from loss of methane, CH4. Alternatively, loss of NH2● could also account for the m/z 78 peak. These fragmentation channels are summarized below in Scheme 1.

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Scheme 1. Fragmentation channels of protonated aniline. Channel 1 is the dominant channel for the N-protonated tautomer. Channels 2–5a are observed for the p-protonated tautomer. The fragment peak at m/z 78 for the p-protonated species could arise from sequential loses of H and CH3 (channels 2 and 3), or from channel 5a. Although possible, isotopic substitution studies suggest that channel 5b is not accessed under the conditions employed in this work.

To further investigate the fragmentation channels, three isotopologues of aniline were also studied: (i)

12

C6D514NH2, (ii)

12

C6H515NH2, and (iii) 13C6H514NH2. The DMS behaviors of these

species were similar to that of the naturally occurring aniline isotopologue, so the N-protonated and p-protonated tautomers could be easily separated and probed individually. The fragmentation spectra for the DMS-selected tautomers of the protonated isotopologues are available in the supporting information that accompanies this manuscript. In the case of the deuterated analogue, (12C6D514NH2 + H)+, the major product channel for the N-protonated tautomer corresponded to production of

14

NH3 (channel 1, see Scheme 1), supporting the assignment. Minor product

channels corresponding to loss of 12CD3 and H12CD3 were also observed, suggesting that proton scrambling during CID produces the p-protonated tautomer and accesses fragmentation channels 3 and 5a (see Scheme 1). The major loss channel for the p-protonated tautomer of (12C6D514NH2 + H)+ is loss of 18 mass units, likely corresponding to production of 14NH2D via channel 4 and 12

CD3 via channel 3. We also observe loss of

14

NH3 via channel 4 and H12CD3 via channel 5a.

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The N-protonated tautomer of the

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N isotopologue produces only

15

NH3 upon fragmentation

(channel 1). This is also the dominant fragmentation pathway for the p-protonated tautomer of (12C6H515NH2 + H)+ (channel 4), but there is some evidence for production of CH3● (channel 3) and CH4 (channel 5a). Both tautomers of the (13C6H514NH2 + H)+ isotopologue lose 17 mass units upon fragmentation. This corresponds to production of

14

NH3 and/or

13

CH4; given the

observations for the other isotopologues studied, we favor product channel 1 for fragmentation of the N-protonated tautomer and a combination of channels 4 and 5a for the p-protonated tautomer. These results are summarized in Table 1. Table 1. The observed fragmentation channels of protonated aniline. 12 12 12 13 Product C6H514NH2 C6D514NH2 C6H515NH2 C6H514NH2 Channel N-prot. p-prot. N-prot. p-prot. N-prot. p-prot. N-prot. p-prot. 1 X X X X 2 X 3 X X X X 4 X X X X 5a X X X X X 5b

Figure 4 shows the dispersion plot recorded for protonated aniline in a pure N2 environment at T = 150 °C. Both of the tautomers exhibit Type B behavior within a pure N2 environment, indicating weak clustering interactions.28, 33, 42 Similar behavior has been observed previously for protonated 4-ABA and for protonated nucleobases.1,

22

In the case of the protonated 4-ABA,

Campbell et al. proposed that the differing dipole moments between the N- and O-protonated tautomers yielded slight differences in interaction with the N2 environment, ultimately leading to the different differential mobilities exhibited by the two species.22 Using similar arguments for protonated aniline, one would expect that the resonance-stabilized p-protonated tautomer should have a smaller dipole moment than the N-protonated species (which is not resonance stabilized), leading to weaker interactions with the N2 transport gas. Based on that reasoning, one would

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expect that the red curve in Figure 4 should be associated with the p-protonated molecule, and that the black curve should be associated with the N-protonated molecule. This assignment is internally consistent with our conclusions from the fragmentation study. To further support this conclusion, high level electronic structure calculations were conducted for the N-protonated and p-protonated tautomers of aniline. Each molecule was first geometry optimized at the B3LYP/ 6311++G(d,p) level of theory, then single point energy calculations were conducted at the CCSD(T)/6-311++G(d,p) level of theory. This yielded dipole moments of 1.2709 D and 7.3065 D for the p-protonated and N-protonated species, respectively, in agreement with expectations based on resonance arguments.

Figure 4. The dispersion plot of protonated aniline, (12C6H514NH2 + H)+, recorded in pure N2 at a pressure of P = 1 atm. The black trace is assigned to the N-protonated tautomer, and the red trace to the p-protonated tautomer. Error bars give the 2σ confidence interval of the mean peak position (three repeat experiments). The green bar highlights the region associated with the ionogram that is shown in Figure 3. Between the DMS cell and the Q-jet region of the DMS-MS instrument (see Figure 1), there is a high-pressure region to which one can add a low partial pressure of hydrogen-deuterium

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exchange (HDX) reagent via the throttle gas. In doing so, one can induce the exchange of labile hydrogen nuclei for deuterium nuclei, and monitor these changes with the mass spectrometer to infer information about the structure of the DMS-selected parent ion.1, 23 For the N-protonated tautomer of aniline, one would expect there should be three labile hydrogen nuclei – those associated with the ammonium group – and so a maximum of three H/D exchanges should be possible. For the p-protonated tautomer, protonation of the ring should result in a maximum of six labile C–H hydrogen atoms (assuming the ring H atoms all become labile upon breaking aromaticity) in addition to the two H atoms associated with the amino group. Thus, one would expect to observe more HDX for the p-protonated tautomer than for the N-protonated tautomer. Figure 5 plots the results of HDX experiments for the DMS-selected N-protonated and pprotonated tautomers of aniline. Intensities in Figure 5 are reported as the difference between the parent-ion-normalized mass spectra with and without the HDX reagent (CH3OD) added to the throttle gas. A single H/D exchange increases the ion mass by one mass unit. Thus, the Nprotonated tautomer (Figure 5; top panel) exhibits three H/D events, with each successive exchange occurring with diminishing probability. The S/N of the HDX difference spectrum of the p-protonated tautomer is somewhat poorer than that of the N-protonated tautomer due to the relatively low signal intensity of the p-protonated ion (Figure 5; bottom panel). Nevertheless, it is clear that the relative rates and extent of HDX is greater for the p-protonated species compared to the N-protonated species. Unfortunately, due to an intense contaminant signal at m/z 100 and 101, we are unable to determine whether all seven labile H atoms of the p-protonated tautomer exchanged. However, we do observe up to five H/D exchanges, which is consistent with our hypothesis of labile ring hydrogen atoms for the p-protonated ion. Interestingly, both the Nprotonated and p-protonated tautomers exhibit loss of ND3 as the sole product channel post-

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HDX. This suggests that the p-protonated tautomer is converted to the N-protonated tautomer by the HDX reagent. A similar scenario was observed in our study of 4-ABA;23 the phenomenon of solvent-vapor-driven tautomer interconversion is discussed in more detail below.

Figure 5. HDX difference spectra for the (top) N-protonated and (bottom) p-protonated tautomers of aniline. Mass spectra for each species were recorded when pure N2 was used for the throttle gas and when the N2 throttle gas was saturated with deuterated methanol vapor, CH3OD. The spectra were normalized to the m/z 94 (parent ion) peak before the difference was taken.

In examining the ionograms of protonated aniline, we find little variation in the relative peak areas for each tautomer as a function of SV, which implies that tautomer interconversion does not occur at the temperatures in the DMS cell within the timescale of the experiment. Peak integration yields relative populations of 96 % N-protonated aniline and 4 % p-protonated aniline within the probed ensemble. These values carry a standard error of ca. 3 %. To estimate the relative population of the various tautomers of protonated aniline as a function of temperature, we conducted a series of Gibbs’ energy calculations using the G3MP2 method as implemented in Gaussian 09.36, 46 At the temperature of the DMS cell (150 °C), our calculations predict relative populations of the N-protonated and p-protonated tautomers to be 97 % and 3 %, respectively

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(see Figure 6). This is within the measured standard deviation of ± 1.5 %. To achieve a population ratio of 96:4, the G3MP2 calculations predict that a temperature of T ≈ 550 K (ca. 277 °C) is required. Note that our calculations are in general agreement with previous computational work, which predict tautomer ratios between 99:1 and 90:10.12, 14, 16

Figure 6. The relative populations of the (blue squares) N-protonated, (red up-triangles) p-protonated, and (green down-triangles) o-protonated tautomers of aniline as a function of temperature as determined with G3MP2 calculations. The experimentally measured relative populations (using acetonitrile for ESI) are plotted as dashed horizontal lines. The transparent blue and green regions highlight the 1σ confidence interval.

Several recent publications have shown that the relative populations of tautomeric species can be heavily influence by choice of the ESI solvent,9, 11, 47 the source conditions,13, 24 and gas phase clustering.23 While we did not observe variations in the N-protonated and p-protonated aniline relative populations as a function of source voltages (viz. field-induced temperature), we could significantly manipulate ensemble sub-populations by varying ESI solvent or solvent partial pressure within the high pressure region of the DMS-MS instrument. Figure 7 plots the fragmentation spectra of the N-protonated and p-protonated tautomers following DMS-selection

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when employing either acetonitrile (ACN) or 50:50 methanol:water (MeOH) as the ESI solvent. In both cases, the N-protonated tautomer dominates the ensemble population (Figure 7A). However, employing a protic solvent for ESI essentially quenches the p-protonated species (Figure 7B). Moreover, the fragmentation spectrum for the p-protonated tautomer also changes when MeOH is substituted for ACN as the ESI solvent, adopting a pattern that is intermediate between the N- and p-protonated patterns observed following ESI from ACN. This suggests that the protic ESI solvent drives tautomerization during and/or post DMS selection, likely by introducing a low partial pressure of solvent vapor into the DMS N2 collision environment.

Figure 7. (A) The fragmentation spectrum observed for N-protonated aniline following ESI from acetonitrile (ACN; blue) and 50:50 water:methanol (MeOH; black, reflected). (B) The fragmentation spectrum observed for p-protonated aniline following ESI from acetonitrile (ACN; blue) and 50:50 water:methanol (MeOH; black, reflected). The intensity of the spectrum of the p-protonated species is plotted relative to that of the Nprotonated species.

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As mentioned above, previous work from our group has shown that gas-phase clustering with protic solvent molecules can induce tautomer interconversion.23 To test whether this is the case for protonated aniline, we repurposed the post-DMS HDX environment as a solvent clustering cell such that we could study the effect of ion-solvent clustering on the fragmentation behavior of the DMS-separated tautomers. To ensure that the N-protonated and p-protonated tautomers were present in the probed ensemble, ACN was used as the ESI solvent and the fragmentation spectra of the two tautomers were recorded following separation in the dry N2 environment. This generated the fragmentation spectra that are typical of the N-protonated and p-protonated tautomers (discussed above). When a low partial pressure of H2O is admitted to the HDX region of the DMS-MS instrument (post DMS-separation) we observe no change in the fragmentation spectrum of the N-protonated ion. However, the fragmentation spectrum of the p-protonated species changes significantly; the m/z 93, 79, and 78 fragment peaks are substantially reduced in relative intensity in favor of the m/z 77 peak (NH3 loss channel). These results are plotted in Figure 8. The N- and p-protonated tautomers of the deuterated analogue, (C6D5NH2 + H)+, were also studied in a similar experiment. As expected, following exposure to H2O vapor, the Nprotonated tautomer showed no change in its fragmentation profile. Interestingly, following exposure to a low partial pressure of water vapor the p-protonated tautomer exhibited significant increases in the intensities of fragments associated with loss of 17 amu, 18 amu, and 19 amu (see Supporting Information). These channels are consistent with loss of NH3, NH2D, and NHD2. This indicates that deuterium scrambling is occurring, likely owing to the increased lability of D due to protonation on the aromatic ring.

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Figure 8. (Top left panel) The fragmentation spectrum observed for N-protonated aniline following exposure to a (red trace) dry N2 environment and (black trace; reflected) an N2 environment saturated with water vapor. (Top right panel) The fragmentation spectrum observed for p-protonated aniline following exposure to a (red trace) dry N2 environment and (black trace; reflected) an N2 environment saturated with water vapor. (Bottom) the lowest energy structures for the N-protonated, waterbridged, and p-protonated isomers of (C6H5NH2 +H)+•(H2O)4. To support this experimental work, we conducted a detailed computational study to determine the relative energies of (C6H5NH2 +H)+•(solv.)n (n = 0 – 5; solv. = H2O, CH3OH). Identifying the lowest energy protonation site computationally has historically been very difficult, with different methods yielding either the N- or p-protonated tautomer as the global minimum structure.6,

18, 24, 48

For example, the B3LYP/6-311++G(d,p) method yields the p-

protonated species as the global minimum structure for bare protonated aniline, 4.2 kJ/mol lower in energy than the N-protonated form (see Figure 9). However, the CCSD(T)/6-311++G(d,p) method identifies the N-protonated species as the lowest energy tautomer by 7.5 kJ/mol. Upon addition of even one solvent molecule, both the DFT and coupled cluster methods favor the Nprotonated tautomer to be lowest in energy. In examining the structures of the ion-solvent

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clusters, we find that the solvent molecules generally prefer to localize near the site of protonation. For example, Figure 8 shows the lowest energy structures for the N- and pprotonated tautomers of (C6H5NH2 +H)+•(H2O)4. Interestingly, when there are more than three solvent molecules present, solvent-bridged structures consisting of hydrogen-bonded solvent networks extending from the N atom to the para-carbon atom are also found by our BH algorithm; Figure 8 also plots the lowest energy solvent-bridged structure for (C6H5NH2 +H)+•(H2O)4. Note that the charge-carrying proton is located in the solvent hydrogen-bonding network of the solvent-bridged species, rather than on the aniline molecule. These structures provide a proton-relay pathway to transfer the charge-carrying proton from the para-carbon atom to the N atom,49-50 similar to those identified in our previous work on 4-aminobenzoic acid.23 Figure 9 compares the calculated relative energies of the three structural motifs for (C6H5NH2 +H)+•(solv.)n (n = 0 – 5; solv. = H2O, CH3OH). These results indicate that the p-protonated tautomer is kinetically trapped during the ESI process, and that it can be “untrapped” by forming clusters with three or more protic solvent molecules in the high-pressure region of the DMS-MS ion source. Following formation, the p-protonated (C6H5NH2 +H)+•(solv.)n (n > 3; solv. = H2O, CH3OH) species can undergo isomerization/annealing in the high-pressure environment of the DMS-MS instrument to sample the lower energy region of the potential energy surface (PES) which is associated with the solvent-bridged structure, thus serving to remove the chargecarrying proton from the para-carbon atom. Evaporation of the solvent molecules from the solvent-bridged structure or further isomerization to produce the global minimum N-protonated isomer (followed by solvent evaporation) would then generate the bare N-protonated aniline tautomer, consistent with experimental observations.

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Figure 9. (Left panel) The relative standard Gibb’s energies of the lowest energy structures for the N-protonated, water-bridged, and p-protonated isomers of (C6H5NH2 +H)+•(H2O)n (n = 0–5). (Right panel) The relative standard Gibb’s energies of the lowest energy structures for the N-protonated, water-bridged, and p-protonated isomers of (C6H5NH2 +H)+•(CH3OH)n (n = 0–5). Solid lines show results for B3LYP/6-311++G(d,p) + GD3 calculations and dashed red lines show the results for CCSD(T)/ 6-311++G(d,p) calculations.

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5. Conclusions This study illustrates the ability of DMS to separate and probe the tautomers of protonated aniline. DMS behavior, HDX rates, and fragmentation spectra indicate that two tautomeric forms of (C6H5NH2 +H)+ are produced by ESI. These results, when taken together with high-level electronic structure calculations, show that the N-protonated form of (C6H5NH2 +H)+ is the major contributor to the ensemble population (~96%), and that only a minor amount of the pprotonated tautomer is present. Clustering experiments conducted on the isolated N- and pprotonated tautomers show that the p-protonated species can be converted to the N-protonated form, but not vice versa. This suggests that the N-protonated tautomer is the global minimum structure, as predicted by calculations at the coupled cluster level of theory. A computational survey of the (C6H5NH2 +H)+•(solv.)n (n = 1–5; solv. = H2O, CH3OH) species shows that there are three structural motifs associated with distinct regions of the cluster potential energy landscape: N-protonated, p-protonated, and solvent-bridged. The N-protonated species are significantly lower in energy that the other isomers in all cases studied here. The solvent-bridged structures are especially interesting since these suggest that, upon reaching a critical size, ion-solvent clusters can tautomerize via a Grotthuss-like mechanism. This phenomenon, which is likely to be important early in the ESI process (if protic solvents are used) and in high pressure HDX experiments, can alter ion structure prior to MS characterization. This further underscores the fact that care should be taken when using mass spectrometric techniques to determine molecular structure.

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Associated Content Supporting Information. Calculated cluster Cartesian atomic coordinates, thermochemical data, and isotopologue fragmentation spectra are provided free of charge. Acknowledgments The authors acknowledge the financial support from the Natural Sciences and Engineering Research Council (NSERC) of Canada and high-performance computing support from the SHARCNET consortium of Compute Canada. They also thank Professor Terry McMahon (University of Waterloo), as well as Drs. Jim Hager, Bradley Schneider, and Yves Le Blanc (SCIEX) for helpful conversations. The computational resources of the Department of Chemistry at the University of the Pacific in Stockton, CA are also gratefully acknowledged (BB).

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16. Nold, M. J.; Wesdemiotis, C. Differentiation of N- from C-protonated aniline by neutralization-reionization. J. Mass Spectrom. 1996, 31, 1169-1172. 17. Pachuta, S. J.; Isernflecha, I.; Cooks, R. G. Charge Stripping and the Site of Cationization of Substituted Aromatic Compounds. Org. Mass Spectrom. 1986, 21, 1-5. 18. Russo, N.; Toscano, M.; Grand, A.; Mineva, T. Proton affinity and protonation sites of aniline. Energetic behavior and density functional reactivity indices. J. Phys. Chem. A 2000, 104, 4017-4021. 19. Wood, K. V.; Burinsky, D. J.; Cameron, D.; Cooks, R. G. Site of Gas-Phase Cation Attachement - Protonation, Methylation, and Ethylation of Aniline, Phenol, and Thiophenol. Journal of Organic Chemistry 1983, 48, 5236-5242. 20. Shvartsburg, A. A.; Smith, R. D. Fundamentals of Traveling Wave Ion Mobility Spectrometry. Anal. Chem. 2008, 80, 9689-9699. 21. Merenbloom, S. I.; Flick, T. G.; Williams, E. R. How Hot are Your Ions in TWAVE Ion Mobility Spectrometry? J. Am. Soc. Mass Spectrom. 2012, 23, 553-562. 22. Campbell, J. L.; Le Blanc, J. C. Y.; Schneider, B. B. Probing Electrospray Ionization Dynamics Using Differential Mobility Spectrometry: The Curious Case of 4-Aminobenzoic Acid. Anal. Chem. 2012, 84, 7857-7864. 23. Campbell, J. L.; Yang, A. M. C.; Melo, L. R.; Hopkins, W. S. Studying Gas-Phase Interconversion of Tautomers Using Differential Mobility Spectrometry. J. Am. Soc. Mass Spectrom. 2016, 27, 1277-1284. 24. Xia, H.; Attygalle, A. B. Untrapping Kinetically Trapped Ions: The Role of Water Vapor and Ion-Source Activation Conditions on the Gas-Phase Protomer Ratio of Benzocaine Revealed by Ion-Mobility Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2017, 28, 2580-2587 25. Collings, B. A.; Romaschin, M. A. MS/MS of Ions in a Low Pressure Linear Ion Trap using a Pulsed Gas. J. Am. Soc. Mass Spectrom. 2009, 20, 1714-1717. 26. Guna, M.; Biesenthal, T. A. Performance Enhancements of Mass Selective Axial Ejection from a Linear Ion Trap. J. Am. Soc. Mass Spectrom. 2009, 20, 1132-1140. 27. Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry. Int. J. Mass Spectrom. 2010, 298, 45-54. 28. Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Chemical Effects in the Separation Process of a Differential Mobility/Mass Spectrometer System. Anal. Chem. 2010, 82, 1867-1880. 29. Eiceman, G. A.; Z., K. Ion mobility spectrometry. 2nd ed.; CRC Press: Boca Raton, 2005. 30. Krylov, E. V.; Nazarov, E. G.; Miller, R. A. Differential mobility spectrometer: Model of operation. Int. J. Mass Spectrom. 2007, 266, 76-85. 31. Purves, R. W.; Guevremont, R. Electrospray ionization high-field asymmetric waveform ion mobility spectrometry-mass spectrometry. Anal. Chem. 1999, 71, 2346-2357. 32. Shvartsburg, A. A. Differential ion mobility spectrometry: nonlinear ion transport and fundamentals of FAIMS. CRC Press: Boca Raton, 2009.

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33. Hopkins, W. S. Determining the properties of gas-phase clusters. Mol. Phys. 2015, 113, 3151-3158. 34. Becke, A. D. Density-Functional Exchange-Energy Approximation with Correct AsymptoticBehavior. Physical Review A 1988, 38, 3098-3100. 35. Becke, A. D. Density-Functional Thermochemistry .3. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648-5652. 36. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; al., e. Gaussian 09 Revision D.01. In Gaussian, Inc. Wallingford CT 2009. 37. Wiberg, K. B.; Rablen, P. R. Comparison of Atomic Charges Derived via Different Procedures. Journal of Computational Chemistry 1993, 14, 1504-1518. 38. Hopkins, W. S.; Marta, R. A.; McMahon, T. B. Proton-Bound 3-Cyanophenylalanine Trimethylamine Clusters: Isomer-Specific Fragmentation Pathways and Evidence of Gas-Phase Zwitterions. J. Phys. Chem. A 2013, 117, 10714-10718. 39. Lecours, M. J.; Chow, W. C. T.; Hopkins, W. S. Density Functional Theory Study of RhnS0,+/- and Rhn+10,+/- (n=1-9). J. Phys. Chem. A 2014, 118, 4278-4287. 40. Wales, D. J. Energy Landscapes. Cambridge University Press: Cambridge, UK, 2003. 41. Wales, D. J.; Doye, J. P. K. Global optimization by basin-hopping and the lowest energy structures of Lennard-Jones clusters containing up to 110 atoms. J. Phys. Chem. A 1997, 101, 5111-5116. 42. Campbell, J. L.; Zhu, M.; Hopkins, W. S. Ion-Molecule Clustering in Differential Mobility Spectrometry: Lessons Learned from Tetraalkylammonium Cations and their Isomers. J. Am. Soc. Mass Spectrom. 2014, 25, 1583-1591. 43. Liu, C.; Le Blanc, J. C. Y.; Schneider, B. B.; Shields, J.; Federico, J. J.; Zhang, H.; Stroh, J. G.; Kauffman, G. W.; Kung, D. W.; Ieritano, C.; Shepherdson, E.; Verbuyst, M.; Melo, L.; Hasan, M.; Naser, D.; Janiszewski, J. S.; Hopkins, W. S.; Campbell, J. L. Assessing Physicochemical Properties of Drug Molecules via Microsolvation Measurements with Differential Mobility Spectrometry. ACS Central Sci. 2017, 3, 101-109. 44. Liu, C.; Le Blanc, J. C. Y.; Shields, J.; Janiszewski, J. S.; Ieritano, C.; Ye, G. F.; Hawes, G. F.; Hopkins, W. S.; Campbell, J. L. Using differential mobility spectrometry to measure ion solvation: an examination of the roles of solvents and ionic structures in separating quinolinebased drugs. Analyst 2015, 14, 6897-6903. 45. Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 2010, 132, 19. 46. Curtiss, L. A.; Redfern, P. C.; Raghavachari, K.; Rassolov, V.; Pople, J. A. Gaussian-3 theory using reduced Moller-Plesset order. J. Chem. Phys. 1999, 110, 4703-4709. 47. Schroder, D.; Budesinsky, M.; Roithova, J. Deprotonation of p-Hydroxybenzoic Acid: Does Electrospray Ionization Sample Solution or Gas-Phase Structures? J. Am. Chem. Soc. 2012, 134, 15897-15905.

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48. Flammang, R.; Dechamps, N.; Pascal, L.; Van Haverbeke, Y.; Gerbaux, P.; Nam, P. C.; Nguyen, M. T. Ring versus nitrogen protonation of anilines. Lett. Org. Chem. 2004, 1, 23-30. 49. Campbell, S.; Rodgers, M. T.; Marzluff, E. M.; Beauchamp, J. L. Deuterium exchange reactions as a probe of biomolecule structure. Fundamental studies of cas phase H/D exchange reactions of protonated glycine oligomers with D2O, CD3OD, CD3CO2D, and ND3. J. Am. Chem. Soc. 1995, 117, 12840-12854. 50. C.J.T., d. G. Sur la decomposition de l'eau et des corps qu'elle tient en dissolution a l'aide de l'electricite galvanique. Ann. Chim. (Paris) 1806, 58, 19.

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C6H5NH3 1 C H NH2 26 6 3

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C6H5 + NH3 +

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