Hydration-induced protomer switching in p -aminobenzoic acid studied by cold double ion trap infrared spectroscopy †

Para -Aminobenzoic acid (PABA) is a benchmark molecule to study solvent-induced proton site switching. Protonation of the carboxy and amino groups of PABA generates O-and N-protomers of PABAH + , respectively. Ion mobility mass spectrometry (IMS) and infrared photodissociation (IRPD) studies have claimed that the O-protomer most stable in the gas phase is converted to the N-protomer most stable in solution upon hydration with six water molecules in the gas-phase cluster. However, the threshold size has remained ambiguous because the arrival time distributions in the IMS experiments exhibit multiple peaks. On the other hand, IRPD spectroscopy could not detect the N-protomer for smaller hydrated clusters because of broad background due to annealing required to reduce kinetic trapping. Herein, we report the threshold size for O - N protomer switching without ambiguity using IR spectroscopy in a double ion trap spectrometer from 1300 to 1800 cm (cid:2) 1 . The pure O-protomer is prepared by electrospray, and size-specific hydrated clusters are formed in a reaction ion trap. The resulting clusters are transferred into a second cryogenic ion trap and the distribution of O-and N-protomers is determined by mid-IR spectroscopy without broadening. The threshold to promote O - N protomer switching is indeed five water molecules. It is smaller than the value reported previously, and as a result, its pentahydrated structure does not support the Grotthuss mechanism proposed previously. The extent of O - N proton transfer is evaluated by collision-assisted stripping IR spectroscopy, and the N-protomer population increases with the number of water molecules. This result is consistent with the dominant population of the N-protomer in aqueous solution.


Introduction
Protonation is one of the most fundamental chemical reactions and a basic step in many synthetic and catalytic processes.2][3][4] In addition, some molecules vary their preferred protonation site depending on the surrounding environment such as solvent or counter ion.  Sucprotonation site-switching obviously has a huge effect on the chemical reactivity and biological activity of the molecule. 12,284][15][16][17][18][20][21][22]29 PABA has two lowenergy protonated isomers, namely the O-and N-protomers resulting from protonation of the carboxy and amino groups, respectively (Fig. 1).Their relative stability varies with type and degree of solvation.While the O-protomer is most stable in the gas phase, the N-protomer becomes more stable in polar solvents such as water.The high stability of the O-protomer in the gas phase arises from the conjugated p-electron system extending from the neutral amino group to the protonated carboxy group.On the other hand, the N-protomer becomes preferable in polar solvents due to its larger solvation energy. 14nfrared (IR) spectroscopy and ion mobility mass spectrometry (IMS) have previously been used to identify the protonation site in polyhydrated clusters of PABAH + .Hebert and Russel  This journal is © the Owner Societies 2023 tracked changes in the protonation positions in PABAH + (H 2 O) n by low-temperature IMS. 30 They used the electrospray source with a mixed solvent of acetonitrile and water to generate simultaneously both O-and N-protomers.Both protomers are clearly separated by the arrival time distribution (ATD) of the monomer.For clusters up to n = 4, they fitted two Gaussian functions to the ATD but changed to a single broader Gaussian function for n = 5 and a narrower Gaussian for n = 6.From these results, they concluded that the protomer population is dominated by the N-protomer at n = 6.To rationalize this result, they proposed a Grotthus mechanism for proton transfer along a water bridge between the NH 2 and COOH groups at n = 6.However, the ATD exhibit for all hydrated PABAH + clusters multiple peaks and fitting these by only one or two Gaussians may not be fully appropriate, although the DFT calculations also show that the N-protomer becomes most stable at n = 6.
In contrast to the less structure-sensitive IMS spectra, IR spectroscopy can provide direct evidence for the structural assignments of the O-and N-protomers.To this end, Williams and co-workers measured the IR photodissociation (IRPD) spectra of PABAH + (H 2 O) n (n = 1-6) in the OH/NH stretch range (2600-3900 cm À1 ) to determine how many water molecules are necessary to stabilize the N-protomer. 10They introduced PABAH + (H 2 O) n clusters generated directly in the electrospray source into the ion cyclotron resonance mass spectrometer and measured their IRPD spectra.It is known that the electrospray of PABA with water solvent selectively generates the O-protomer of the PABAH + monomer.The IRPD spectra of PABAH + (H 2 O) n (n = 1-5) give free NH stretching bands similar to the monomer, while new vibrational bands appear with high intensity in the spectrum of n = 6 (although such bands are already weakly present also for n o 6).They assigned the new bands to the N-protomer in the hydrated cluster, and concluded that hydration by six water molecules stabilizes the N-protomer such that the preferred protonation site switches from COOH to NH 2 in PABAH + at n = 6.One problem of this approach is kinetic trapping of the N-protomer in the hydrated cluster in the electrospray process.As the N-protomer is more stable in aqueous solution, it may remain in the hydrated clusters generated in the electrospray source regardless of its relative stability.To avoid the kinetically trapped N-protomer, they carefully annealed the hydrated clusters by collisions with N 2 and measured the IRPD spectra at 130 K.Although annealing and the measurement at 130 K were assumed to avoid kinetic trapping, all IRPD spectra became broader and the spectral signatures of the two protomers became less resolved.Consequently, the presence of the N-protomer in the n = 1-5 spectra is not clear because of the broad background, as stated by the authors.In summary, it appears clear that hydration by six water molecules is sufficient to induce O -N protonation site-switching, however, the minimal number of water molecules required to promote the switching is still unclear.
To reliably determine the threshold for hydration-induced O -N protonation site-switching, we apply herein a more sophisticated spectroscopic strategy.It has clearly been established that only the O-protomer is produced when the PABAH + monomer is generated from the electrospray of a slightly acidic protic solution. 12This O-protomer of the PABAH + monomer is mass-selected and then introduced into the first ion trap (reaction trap 31,32 ) containing water vapour to grow PABAH + (H 2 O) n clusters.If then the N-protomer is detected in the hydrated clusters after the first ion trap by IRPD, it is without any doubt the result of O-N protonation site-switching.The reaction trap has to be kept at a certain elevated temperature (80 K in this work) to promote the intracluster proton transfer reaction.If we measure the IR spectra under such conditions, spectral broadening may not be avoided.This problem is solved by the following means.We use one more ion trap [33][34][35] at very low temperature (4 K in this work) and the hydrated clusters in the warmer reaction trap are mass-selected and transferred into the cryogenic second ion trap at a frozen N/O population ratio.Then, we measure the population of the O-and N-protomers using IRPD spectroscopy by irradiating the mass-selected clusters with a tuneable IR laser (using the tagging approach 36 ).The broadening of IR spectra can be avoided because of the low temperature.In addition, we expand the spectral range down to mid-IR to readily distinguish between the O-and N-protomers.Recently, Johnson and coworkers reported the IRPD spectra of the O-and N-protomers of the PABAH + monomer and found the clear discrimination of both monomers by probing the CQO stretching range (the CQO band can occur only for the Nprotomer). 20In addition, the shape of the CQO stretching band is rather insensitive to H-bonding with water molecules.This is in stark contrast to the previously investigated N-H stretching range, and thus the new IRPD spectra recorded in the mid-IR (6 mm) range will give clear evidence for the protomer assignments.Finally, we also apply the recently developed collision-assisted stripping IR spectroscopic technique (CAS-IR), 27 which enables us to precisely determine the protomer population in highly hydrated clusters.By using both IRPD and CAS-IR in the double ion trap spectrometer, we examine how the protomer abundance ratio in PABAH + changes as the number of water molecules increases.

Experimental and computational methods
A methanol solution of PABA (Wako, 10 À5 M) with 0.5% of formic acid is electrosprayed and the fine droplets are Molecular structures and vibrational frequencies of PABAH + (H 2 O) n are calculated by density functional theory (DFT).The initial structures of PABAH + (H 2 O) n are generated automatically by the OPLS 38 force field implemented in MacroModel. 39Preliminary DFT calculations are performed for the initial structures at the dispersion-corrected oB97X-D/ 6-31G(d,p) level using the Gaussian16 40 software.The oB97X-D method gives better results than B3LYP and MP2. 10,27Relative Gibbs free energies at 298.15 K are also calculated at the same level.The structures whose relative Gibbs free energy are within 10 kJ mol À1 of the most stable protomer are re-calculated at the higher oB97X-D/6-311++G(d,p) level 27 (ESI †).All calculated conformers displayed are local minima on the potential energy surface.The harmonic vibrational frequencies obtained are scaled by 0.952 in the 3 mm range 27 and 0.970 (O-protomer)/ 0.955 (N-protomer) in the 6 mm range.The scaling factor for the 6 mm range is determined by the characteristic band for each protomer at n = 0 (Fig. S1, ESI †).In addition, the Gibbs free energies are re-calculated at 80 K at the higher computational level (Tables S1-S8, ESI †).bands characteristic of the N-protomer of the PABAH + monomer, i.e., the CQO stretch (1790 cm À1 ) and NH 3 umbrella (1470 cm À1 ) vibrations indicated by broken lines, are not observed in the spectrum.Thus, all bands present in the recorded spectrum of PABAH + (n = 0) are attributed to the Oprotomer (color-coded in red).This result indicates that the initial protomeric ion injected into the reaction trap is the most stable gas-phase ion, and kinetic trapping of the higher energy N-protomer most stable in solution is not observed.The IRPD spectra of PABAH + (H 2 O) n (n = 1-7) also show the vibrational bands in the 1500-1700 cm À1 range, which are tentatively assigned to hydrated clusters with an O-protomeric PABAH + core.The NH 3 umbrella band, which is the characteristic mode of the N-PABAH + core, will be shifted in its hydrated clusters because of H-bonding (NHÁ Á ÁO ionic H-bonds).On the other hand, the CQO stretch band is relatively insensitive to hydration because water molecules form H-bonds mainly around the positively charged NH 3 + centre.Thus, the absence and presence  3a).The bands observed between 1500 and 1650 cm À1 are similar to those in the spectrum computed for the most stable O-protomer in PABAH + (H 2 O) 4 .The theoretical spectra for the N-protomer predict the CQO stretch band at 1765 cm À1 .However, no clear band is observed in the range above 1700 cm À1 , suggesting that the population of the N-protomer in PABAH + (H 2 O) 4 is (at most) small and thus below the detection limit (Fig. S6-S9, ESI †).This observation is indeed consistent with the calculated Gibbs free energies (80 K) of PABAH + (H 2 O) 4 , for which the most stable isomer with an Nprotomer core is less stable than the most stable one with an Oprotomer core by 12.1 kJ mol À1 .It should be noted that the NH 3 umbrella band for n = 4 is blue-shifted by B90 cm À1 (frequency: 1547 cm À1 , IR intensity: 114 km mol À1 ) and overlaps with bands of the O-protomer, making this vibration insensitive for protomer assignment.

Results and discussion
In contrast to n r 4, the IRPD spectrum of PABAH + (H 2 O) 5 is not solely assigned to the O-protomer.Specifically, the band at 1748 cm À1 cannot be reproduced by O-protomers (Fig. S10 and S12, ESI †).In contrast, one of the theoretical IR spectra of the two most stable pentahydrated PABAH + clusters with an Nprotomer core, for which the CQO stretches are calculated at 1730 and 1768 cm À1 (Fig. 3d), explain the band at 1748 cm À1 (Fig. 3c, d and Fig. S11, S13, ESI †).A more detailed isomer assignment for n = 5 will be given below after assigning the spectra for n = 6 and 7.In any case, the observed band at 1748 cm À1 is a clear signature of the N-protomer core in pentahydrated PABAH + .Bands around 1630 cm À1 are attributed to the most stable O-protomer in pentahydrated clusters.The observation of N-protomer clusters at n = 5 is consistent with their significant stabilization in Gibbs free energy relative to the most stable O-protomer (only 3.5 and 6.9 kJ mol À1 less stable at 80 K) by adding one more water (n = 4 -5).From this assignment, we conclude that the N-protomeric core begins to appear already at n = 5 and not at n = 6 under the current experimental conditions.
The coexistence of O-and N-protomers in the PABAH + (H 2 O) n clusters with n = 6 and 7 is also confirmed by their IRPD spectra shown in Fig. 4a-d, respectively.In both spectra, two vibrational bands are observed in the 1700-1800 cm À1 range, which cannot be assigned to O-protomers (Fig. 4 and Fig. S14, S16,  ESI †).The most stable conformer of PABAH + (H 2 O) 6 has an Nprotomeric core, in agreement with previous calculations. 10he bands at 1726 and 1748 cm À1 can be assigned to CQO stretch modes of the most stable and less stable N-protomers of PABAH + (H 2 O) 6 (Fig. 4a ).It should be noted that the detection sensitivity decreases in the lower frequency range because of the lower dissociation efficiency of H 2 -tagged molecules. 45The calculated intensity of the band at 1638 cm À1 of the O-protomer core is 686 km mol À1 .
View Article Online group.We name herein the conformers with and without Hbonding between CQO and water as bridged and unbridged conformers, respectively.Similar to n = 6, two CQO stretch bands appear at n = 7, but their relative intensities are reversed, indicating that the preferred geometry for n = 7 is a bridged conformer (Fig. 4c, d and Fig. S18-S21, ESI †).Comparing the energies of the two types of structures for each hydrated cluster, the bridged structure is more stabilized in n = 7 than in n = 6 (by 12.8 vs. 7.2 kJ mol À1 ).
When we now look at the single CQO stretch band observed for n = 5, it can be assigned to an unbridged conformer by comparing its frequency to those for n = 6 and 7.However, this type of CQO stretch is calculated for a metastable conformer, which is 3.4 kJ mol À1 less stable than the most stable Nprotomer (Fig. 3 and Fig. S13, ESI †).A similar inconsistency is found for n = 6.The higher-frequency CQO stretch band has to be assigned to the unbridged conformer which is more than 7 kJ mol À1 less stable than the most stable bridged one (Fig. S15, ESI †), although the band of the unbridged isomer is more intense than the one of the bridged isomer.The most probable scenario is that the reaction product, the N-protomer, is thermally not equilibrated at 80 K.The produced N-protomer is probably hotter than 80 K because of exothermic intracluster O -N proton transfer.Alternatively, hot O-protomers may transiently be generated by hydration of PABAH + by converting hydration energy into internal energy.As a result, the hot Oprotomer may overcome the reaction barrier for O -N proton transfer.The produced ''hot'' N-protomer may then be kinetically trapped in the cold reaction trap (80 K).This means that the kinetically trapped N-protomer reflects the structural information at higher temperature.Therefore, the energy gap between experiment and theory arises from a temperature effect.In fact, when we elevate the temperature from 80 to 298 K for the free energy calculations, unbridged N-protomers become more stable than bridged ones for n = 5 and 6 (Fig. S22, ESI †), qualitatively consistent with the experimental IRPD spectra.The higher stability of the unbridged N-protomers at higher temperature can then be ascribed to entropy.
Although the IRPD spectra shown in Fig. 3 and 4 clearly indicate the presence/absence of the N-protomer, they do not directly reveal the population ratio of O-and N-protomers.The population ratio may be derived by normalizing the integrated experimental band intensities using the computed IR oscillator strengths, which however can vary strongly among the various conformers.The exact conformer assignments are very difficult because there are too many possible conformers predicted in the low energy range (e.g., more than ten conformers are lower than 3 kJ mol À1 for the n = 6 clusters of the O-protomer core).This makes a reliable population analysis almost impossible.To avoid this problem, collision-stripping assisted IR (CAS-IR) spectroscopy has been developed and tested for a prototypical molecule, benzocaine H + , an analogue of PABAH + . 27This method measures the populations of O-and N-protomers after stripping all water molecules from the protonated solute molecule and thus the conformational distributions in the hydrated clusters must not necessary be determined.Significantly, the collisional stripping does not affect the protonation position. 27ere, we apply CAS-IR spectroscopy to PABAH + (H 2 O) n and experimentally determine the relative abundance of O-and Nprotomers for each size of the hydrated clusters.
To this end, Fig. 5c shows CAS-IR spectra of PABAH + (H 2 O) n (n = 0-7).After stripping the water ligands, PABAH + forms van der Waals complexes with H 2 , which is introduced into the second ion trap together with the He buffer gas.Then, the IR transitions of the PABAH + monomer can be measured by loss of H 2 (tagging method 36 ).Thus, all bands can be assigned by vibrational transitions of the monomer isomers.The spectrum for n = 0 shows four bands in the 3400-3600 cm À1 range.These bands are in good agreement with the calculated spectrum of O-protonated PABAH + tagged by four H 2 molecules (Fig. 5 and Fig. S23, ESI †).The bands at 3435, 3496, 3535, and 3558 cm À1 are assigned to symmetric NH stretching, outward-facing OH stretching, antisymmetric NH stretching, and OH stretching toward the benzene ring, respectively.Only these bands are observed for mono-to tetrahydrated PABAH + clusters after collisional stripping of the water ligands.Thus, PABAH + has only the O-protonated form in these clusters with one to four water molecules.
In the CAS-IR spectrum of n = 5, new bands appear in the range from 3200 to 3350 cm À1 .From the comparison to the theoretical IR spectra shown in Fig. 5b, these bands at 3230, 3278, and 3303 cm À1 are assigned to symmetric and two antisymmetric stretching vibrations of the NH 3 group in the N-protomer tagged with four H 2 molecules.The theoretical spectra also predict the OH stretching vibration at 3580 cm À1 .Probably this mode corresponds to the band at 3555 cm À1 , which overlaps with the one of the OH stretching of the Oprotomer, because this band appears more strongly than predicted for the O-protomer.Thus, the appearance of the bands between 3200 and 3350 cm À1 confirms the formation of the Nprotomer in the pentahydrated PABAH + cluster, as well as the interpretation of the IRPD spectra in the 6 mm range.Similarly, the coexistence of O-and N-protomers in the PABAH + (H 2 O) n with n = 6 and 7 is confirmed by the appearance of the bands in the 3200-3350 cm À1 range.
The population ratio of N-and O-protomers is quantitatively estimated by normalizing the CAS-IR band intensities using the calculated IR oscillator strength of PABAH + (more exactly H 2tagged PABAH + ).The CAS-IR intensities are obtained by fitting the CAS-IR spectra with Lorentzian profiles (details are shown in Fig. S24, ESI †).Finally, we obtain the N : O ratio of 6 : 4 for n = 5, 2 : 1 for n = 6, and 3 : 1 for n = 7, indicating that this ratio increases with the number of water molecules (Fig. 5d).It should be noted that the N-protomer is likely to be kinetically trapped and the N 2 O interconversion is not thermally equilibrated.Therefore, the obtained population ratio does not reflect the thermodynamic stability but the extent of the O -N proton transfer reaction.The O -N intracluster proton transfer is more pronounced as the number of attached water ligands is increased in the cluster.This observation suggests that the reaction barrier for the proton transfer is lowered by sequential addition of water molecules.

Summary
We have applied double ion trap IRPD and CAS-IR spectroscopy to hydrated clusters of PABAH + .By selective formation of the O-protomer in the electrospray source and the sequential cluster formation with water molecules in the reaction trap combined with mid-IR spectroscopy in the CQO stretch range, we have determined the threshold size for hydration-induced N-protomer formation as five water molecules, without ambiguity caused by kinetic trapping in the electrospray source.This threshold size of n = 5 is smaller than n = 6 reported by previous studies.Hebert and Russel proposed the Grotthuss mechanism for the intracluster proton transfer in the hydrated clusters of PABAH + because of the bridged structure of the hydrated Nprotomer (n = 6) in which the NH 3 + and COOH groups are connected by a H-bonded water chain. 30However, the bridged conformer appears minor in the current IRPD spectrum for n = 6 and the pentahydrated N-protomer of PABAH + does not have such a bridged structure.Thus, we should consider a non-Grotthuss mechanism for the proton transfer, such as the vehicle mechanism, in this size regime. 19,41,42On the other hand, the bridged structure becomes most abundant in higher hydrated clusters (n = 7).This suggests that the Grotthuss mechanism 13,26,43,44 may also be involved in the proton transfer, especially in larger hydrated clusters.However, a clear conclusion that can be drawn here for the smaller clusters (n r 6) is that if the Grotthuss mechanism plays a role in the proton transfer, it would first require the formation of energetically highly unfavourable structures.The proton transfer mechanism can be further examined in the future by hydration with heavy water (D 2 O).Concerning the N/O protomer population, our measurements are currently limited up to the hydration with seven water molecules.It may be interesting to explore how many water molecules are required to reach a 100% population of the N-protomer as observed in solution.
Another interesting aspect is the acceleration of the proton transfer reaction by raising the temperature of the reaction trap.These future plans, as well as the application of double ion trap spectroscopy 31,32 to other molecules that contain two or more protonation sites, will allow us to explore the mechanism of intracluster proton transfer in hydrated clusters and aqueous solution at the molecular level.

Fig. 2
Fig. 2 shows the IRPD spectra of PABAH + (H 2 O) nr7 recorded in the 1300-1800 cm À1 range.Based on previous studies of bare PABAH + , 20 the bands observed in the IRPD spectrum are assigned to C(OH) asym of a band above 1700 cm À1 can be attributed to the O-and Nprotomer, respectively.This argument means that only Oprotomers are populated in the hydrated PABAH + (H 2 O) n up to four water molecules.For the hydrated cluster with five water molecules, a new band is observed in the range above 1700 cm À1 .This band at 1748 cm À1 is assigned to the CQO stretch of the N-protomeric core, and thus strongly suggests that the hydration by five water molecules makes the Nprotomer of PABAH + stable enough to be detected.Similarly, the IRPD spectra of hydrated clusters with six and seven water molecules show vibrational bands in the range above 1700 cm À1 (1726 and 1748 cm À1 , 1725 and 1746 cm À1 , respectively).Thus, O-and N-protomers coexist in the PABAH + (H 2 O) 5-7 size range.To confirm the presence of the N-protomer, theoretical spectra of O-and N-protomers in PABAH + -(H 2 O) n (n = 0-7) obtained by quantum chemical calculations are compared to the observed spectra.Gibbs free energies of optimized structures for n = 0-7 are listed in Tables S1-S8 in ESI, † respectively.The computed spectra of the ten most stable structures for n = 0-3 are compared to the observed ones in Fig.S2-S5 (ESI †), respectively.The bands observed for n = 0-3 are well assigned to O-protomer transitions (Fig. S2-S5, ESI †).Fig. 3b compares the computed spectra of the most stable N-and O-protonated PABAH + (H 2 O) 4 clusters to the observed IRPD spectrum (Fig.

Fig. 3
Fig. 3 IRPD spectrum of PABAH + (H 2 O) n with (a) n = 4 and (c) n = 5 in comparison with (b,d) theoretical IR spectra of the most stable N-and Oprotomers.Molecular structures are shown next to the computed IR spectra with their relative Gibbs free energies at 80 K in parentheses (in kJ mol À1 ).It should be noted that the detection sensitivity decreases toward the lower frequency range because of the lower dissociation efficiency of H 2 -tagged molecules. 45Calculated intensities of the bands at 1316, 1318, and 1589 cm À1 in the n = 4 cluster of the O-protomer core and 1314 and 1584 cm À1 in the n = 5 cluster of the O-protomer core are 711, 734, 896, 1392, and 863 km mol À1 , respectively.

+Fig. 4
Fig. 4 IRPD spectrum PABAH + (H 2 O) n with (a) n = 6 and (c) n = 7 in comparison with (b and d) theoretical IR spectra of the most stable N-and Oprotomers.Molecular structures are shown next to the computed IR spectra with their relative Gibbs free energies at 80 K in parentheses (in kJ mol À1).It should be noted that the detection sensitivity decreases in the lower frequency range because of the lower dissociation efficiency of H 2 -tagged molecules.45The calculated intensity of the band at 1638 cm À1 of the O-protomer core is 686 km mol À1 .

Fig. 5
Fig. 5 Calculated IR spectra of the (a) O-and (b) N-protomers of PABAH + (H 2 ) 4 .(c) Observed CAS-IRPD spectra of PABAH + (H 2 O) n (n = 0-7) and (d) estimated population ratio for N-and O-protomers for n = 5-7.The calculated frequencies of the OH stretching modes are overestimated compared to the NH stretching modes.