Volume 25
Number 6
14 February 2023
Pages 4363–5294

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Physical Chemistry Chemical Physics
rsc.li/pccp

ISSN 1463-9076

PAPER
Otto Dopfer, Shun-ichi Ishiuchi, Masaaki Fujii et al.
Hydration-induced protomer switching in p -aminobenzoic
acid studied by cold double ion trap infrared spectroscopy

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PAPER

Cite this: Phys. Chem. Chem. Phys.,2023, 25, 4481

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Hydration-induced protomer switching in
p-aminobenzoic acid studied by cold double ion
trap infrared spectroscopy†
Kyota Akasaka,ab Keisuke Hirata, acd Fuad Haddad,e Otto Dopfer,
Shun-ichi Ishiuchi *acd and Masaaki Fujii *abd*dePara-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 moststable in solution upon hydration with six water molecules in the gas-phase cluster. However, thethreshold 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 forsmaller 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 1. The pure O-protomer isprepared 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 Nprotomers is determined by mid-IR spectroscopy without broadening. The threshold to promote O - N
Received 27th September 2022,
Accepted 3rd December 2022
DOI: 10.1039/d2cp04497h

protomer switching is indeed five water molecules. It is smaller than the value reported previously, andas 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, andthe N-protomer population increases with the number of water molecules. This result is consistent withrsc.li/pccp

the dominant population of the N-protomer in aqueous solution.

1. Introduction
Protonation is one of the most fundamental chemical reactions
and a basic step in many synthetic and catalytic processes.
Determining the protonation site is important because itchanges the chemical properties of the molecule, such as redox
behaviour, optical activity, and hydrophobicity.1–4 In addition,aLaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo
Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama226-8503,
Japan. E-mail: ishiuchi.s.aa@m.titech.ac.jp, mfujii@res.titech.ac.jpbSchool of Life Science and Technology, Tokyo Institute of Technology, 4259
Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8503, Japanc
Department of Chemistry, School of Science, Tokyo Institute of Technology, 2-12-1
Ookayama, Meguro-ku, Tokyo 152-8550, JapandInternational Research Frontiers Initiative, Tokyo Institute of Technology, 4259
Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.
E-mail: dopfer@physik.tu-berlin.deeInstitut für Optik und Atomare Physik, Technische Universität Berlin,
Hardenbergstrasse 36, 10623 Berlin, Germany† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d2cp04497h

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some molecules vary their preferred protonation site depending on the surrounding environment such as solvent or counterion.5–29 Such protonation site-switching obviously has a huge
effect on the chemical reactivity and biological activity of themolecule.12,28
Para-aminobenzoic acid (PABA) is a benchmark molecule for
protonation site-switching.8–11,13–18,20–22,29 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 anddegree of solvation. While the O-protomer is most stable in thegas 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 protonatedcarboxy group. On the other hand, the N-protomer becomes
preferable in polar solvents due to its larger solvation energy.14
Infrared (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

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Paper

Fig. 1

Molecular structure of O- and N-protomers of PABAH+.

tracked changes in the protonation positions in PABAH+(H2O)nby low-temperature IMS.30 They used the electrospray sourcewith a mixed solvent of acetonitrile and water to generate
simultaneously both O- and N-protomers. Both protomers areclearly separated by the arrival time distribution (ATD) of themonomer. For clusters up to n = 4, they fitted two Gaussianfunctions to the ATD but changed to a single broader Gaussianfunction for n = 5 and a narrower Gaussian for n = 6. From theseresults, 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 awater bridge between the NH2 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+(H2O)n (n = 1–6) in the OH/NH stretch range(2600–3900 cm 1) to determine how many water molecules arenecessary to stabilize the N-protomer.10 They introduced
PABAH+(H2O)n clusters generated directly in the electrospraysource into the ion cyclotron resonance mass spectrometer andmeasured their IRPD spectra. It is known that the electrosprayof PABA with water solvent selectively generates the O-protomerof the PABAH+ monomer. The IRPD spectra of PABAH+(H2O)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 inthe hydrated cluster, and concluded that hydration by six watermolecules stabilizes the N-protomer such that the preferred protonation site switches from COOH to NH2 in PABAH+ at n = 6. One
problem of this approach is kinetic trapping of the N-protomer inthe 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, theycarefully annealed the hydrated clusters by collisions with N2 andmeasured the IRPD spectra at 130 K. Although annealing and themeasurement at 130 K were assumed to avoid kinetic trapping, all4482 | Phys. Chem. Chem. Phys., 2023, 25, 4481–4488

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IRPD spectra became broader and the spectral signatures of thetwo protomers became less resolved. Consequently, the presenceof the N-protomer in the n = 1–5 spectra is not clear because of thebroad 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 moresophisticated 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.12 This O-protomer of the PABAH+ monomer ismass-selected and then introduced into the first ion trap
(reaction trap31,32) containing water vapour to grow PABAH+(H2O)n clusters. If then the N-protomer is detected in thehydrated 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 solvedby the following means. We use one more ion trap33–35 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 approach36). The
broadening of IR spectra can be avoided because of the lowtemperature. 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 theclear discrimination of both monomers by probing the CQO
stretching range (the CQO band can occur only for the Nprotomer).20 In 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–Hstretching 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 (CASIR),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 thenumber of water molecules increases.

2. Experimental andcomputational methods
A methanol solution of PABA (Wako, 10 5 M) with 0.5% of
formic acid is electrosprayed and the fine droplets areThis journal is © the Owner Societies 2023

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Paper

desolvated in a glass capillary heated to B80 1C. The generated
ions are introduced into the vacuum via an ion funnel. The ionsof interest are mass-selected by a quadrupole mass spectrometer (QMS) and transported by a tapered hexapole ion guide.
The ions are hydrated in an octupole ion trap (reaction trap31,32)
with water vapour introduced by a pulsed valve. The temperature of the reaction trap is maintained at 80 K. Hydrated ions
are mass-selected in the second QMS and introduced into thecryogenic quadrupole ion trap (QIT37) via a quadrupole ion
deflector and an octupole ion guide. The QIT is maintained at
4 K by a closed-cycle two-stage He refrigerator. Helium buﬀer
gas mixed with H2 is introduced into the QIT by a pulsed valve
for collisional cooling of hydrated ions down to B10 K, andsubsequently H2 molecules are attached to the hydrated
PABAH+ cluster (H2 tagging method36). The cold hydrated ions,
PABAH+(H2O)n(H2)m, are irradiated with a tuneable OPO/OPA
IR laser. The PABAH+(H2O)n photofragments produced by the
dissociation of all H2 molecules are monitored by a time-offlight mass spectrometer. H2 is weakly bound so that thestructure of the hydrated PABAH+(H2O)n clusters are not
perturbed.36 All IRPD spectra are measured by monitoring thefragment ion yield generated by H2 dissociation as a function oflaser wavenumber.
When CAS-IR spectra are measured, hydrated ions are
directed into the QIT with high kinetic energy (B16 eV) bylowering the oﬀset voltage at the QIT entrance electrode. The
ions are then collisionally dissociated to bare PABAH+, and H2
molecules are attached to PABAH+. The H2-detached photofragments are detected by scanning the wavenumber of the IR laser.

Fig. 2

Molecular structures and vibrational frequencies of
PABAH+(H2O)n are calculated by density functional theory(DFT). The initial structures of PABAH+(H2O)n are generated
automatically by the OPLS38 force field implemented in
MacroModel.39 Preliminary DFT calculations are performed
for the initial structures at the dispersion-corrected oB97X-D/
6-31G(d,p) level using the Gaussian1640 software. The oB97X-D
method gives better results than B3LYP and MP2.10,27 Relative
Gibbs free energies at 298.15 K are also calculated at the samelevel. The structures whose relative Gibbs free energy are within
10 kJ mol 1 of the most stable protomer are re-calculated at thehigher oB97X-D/6-311++G(d,p) level27 (ESI†). All calculated
conformers displayed are local minima on the potential energy
surface. The harmonic vibrational frequencies obtained arescaled by 0.952 in the 3 mm range27 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 freeenergies are re-calculated at 80 K at the higher computational
level (Tables S1–S8, ESI†).

3. Results and discussion
Fig. 2 shows the IRPD spectra of PABAH+(H2O)nr7 recorded in
the 1300–1800 cm 1 range. Based on previous studies of bare
PABAH+,20 the bands observed in the IRPD spectrum areassigned to C(OH)asym
at 1508 cm 1, C–NHarylat 1519 cm 1,
2
2
aryl
1
bend
C–C(OH)2 at 1570–1600 cm , and NH2
at 1658 cm 1. The

IRPD spectra of PABAH+(H2O)n with n = 0–7.

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Paperbands characteristic of the N-protomer of the PABAH+ monomer, i.e., the CQO stretch (1790 cm 1) and NH3 umbrella(1470 cm 1) vibrations indicated by broken lines, are notobserved in the spectrum. Thus, all bands present in therecorded spectrum of PABAH+ (n = 0) are attributed to the Oprotomer (color-coded in red). This result indicates that theinitial protomeric ion injected into the reaction trap is the moststable gas-phase ion, and kinetic trapping of the higher energy
N-protomer most stable in solution is not observed. The IRPD
spectra of PABAH+(H2O)n (n = 1–7) also show the vibrational
bands in the 1500–1700 cm 1 range, which are tentatively
assigned to hydrated clusters with an O-protomeric PABAH+
core. The NH3 umbrella band, which is the characteristic modeof the N-PABAH+ core, will be shifted in its hydrated clusters
because of H-bonding (NH  O ionic H-bonds). On the other
hand, the CQO stretch band is relatively insensitive to hydration because water molecules form H-bonds mainly around thepositively charged NH3+ centre. Thus, the absence and presenceof a band above 1700 cm 1 can be attributed to the O- and Nprotomer, respectively. This argument means that only Oprotomers are populated in the hydrated PABAH+(H2O)n up to
four water molecules. For the hydrated cluster with five watermolecules, a new band is observed in the range above
1700 cm 1. This band at 1748 cm 1 is assigned to the CQO
stretch of the N-protomeric core, and thus strongly suggests

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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 sevenwater molecules show vibrational bands in the range above
1700 cm 1 (1726 and 1748 cm 1, 1725 and 1746 cm 1, respectively). Thus, O- and N-protomers coexist in the PABAH+(H2O)5–7
size range.
To confirm the presence of the N-protomer, theoretical
spectra of O- and N-protomers in PABAH+-(H2O)n (n = 0–7)
obtained by quantum chemical calculations are compared tothe 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 thecomputed spectra of the most stable N- and O-protonated
PABAH+(H2O)4 clusters to the observed IRPD spectrum(Fig. 3a). The bands observed between 1500 and 1650 cm 1
are similar to those in the spectrum computed for the moststable O-protomer in PABAH+(H2O)4. The theoretical spectra for
the N-protomer predict the CQO stretch band at 1765 cm 1.
However, no clear band is observed in the range above
1700 cm 1, suggesting that the population of the N-protomer
in PABAH+(H2O)4 is (at most) small and thus below theFig. 3 IRPD spectrum of PABAH+(H2O)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 1). It
should be noted that the detection sensitivity decreases toward the lower frequency range because of the lower dissociation eﬃciency of H2-tagged
molecules.45 Calculated intensities of the bands at 1316, 1318, and 1589 cm 1 in the n = 4 cluster of the O-protomer core and 1314 and 1584 cm 1 in the
n = 5 cluster of the O-protomer core are 711, 734, 896, 1392, and 863 km mol 1, respectively.

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detection limit (Fig. S6–S9, ESI†). This observation is indeed
consistent with the calculated Gibbs free energies (80 K) of
PABAH+(H2O)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 1. It should be noted that the NH3umbrella band for n = 4 is blue-shifted by B90 cm 1 (frequency: 1547 cm 1, IR intensity: 114 km mol 1) and overlaps
with bands of the O-protomer, making this vibration insensitive for protomer assignment.
In contrast to n r 4, the IRPD spectrum of PABAH+(H2O)5 is
not solely assigned to the O-protomer. Specifically, the band at
1748 cm 1 cannot be reproduced by O-protomers (Fig. S10 and
S12, ESI†). In contrast, one of the theoretical IR spectra of thetwo most stable pentahydrated PABAH+ clusters with an Nprotomer core, for which the CQO stretches are calculated at1730 and 1768 cm 1 (Fig. 3d), can explain the band at
1748 cm 1 (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 1 is a clear signature of the N-protomer core inpentahydrated PABAH+. Bands around 1630 cm 1 are attributed to the most stable O-protomer in pentahydrated clusters.
The observation of N-protomer clusters at n = 5 is consistentPaperwith their significant stabilization in Gibbs free energy relative
to the most stable O-protomer (only 3.5 and 6.9 kJ mol 1 lessstable at 80 K) by adding one more water (n = 4 - 5). From thisassignment, we conclude that the N-protomeric core begins toappear already at n = 5 and not at n = 6 under the current
experimental conditions.
The coexistence of O- and N-protomers in the PABAH+(H2O)nclusters 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 1 range, whichcannot be assigned to O-protomers (Fig. 4 and Fig. S14, S16,
ESI†). The most stable conformer of PABAH+(H2O)6 has an Nprotomeric core, in agreement with previous calculations.10
The bands at 1726 and 1748 cm 1 can be assigned to CQO
stretch modes of the most stable and less stable N-protomers of
PABAH+(H2O)6 (Fig. 4a, b and Fig. S15, ESI†). The different
frequencies of the two CQO stretch modes arise from thepresence and absence of H-bonding to a water molecule (seecorresponding structures in Fig. 4b and Fig. S17, ESI†). It
should be noted that the free CQO stretching band
(1748 cm 1) in the less stable conformer is slightly redshifted from that of bare PABAH+ (1781 cm 1) probably due
to the change of electron density upon hydration of the NH3+

Fig. 4 IRPD spectrum PABAH+(H2O)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 1). It
should be noted that the detection sensitivity decreases in the lower frequency range because of the lower dissociation eﬃciency of H2-tagged
molecules.45 The calculated intensity of the band at 1638 cm 1 of the O-protomer core is 686 km mol 1.

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Papergroup. 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 theenergies of the two types of structures for each hydrated cluster,
the bridged structure is more stabilized in n = 7 than in n = 6 (by12.8 vs. 7.2 kJ mol 1).
When we now look at the single CQO stretch band observed
for n = 5, it can be assigned to an unbridged conformer bycomparing its frequency to those for n = 6 and 7. However, thistype of CQO stretch is calculated for a metastable conformer,
which is 3.4 kJ mol 1 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 1 less stable than the most stable bridged one(Fig. S15, ESI†), although the band of the unbridged isomer ismore intense than the one of the bridged isomer. The mostprobable 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 protontransfer. The produced ‘‘hot’’ N-protomer may then be kinetically trapped in the cold reaction trap (80 K). This means thatthe kinetically trapped N-protomer reflects the structural information at higher temperature. Therefore, the energy gap

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between experiment and theory arises from a temperatureeﬀect. In fact, when we elevate the temperature from 80 to298 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 notdirectly 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 1 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 prototypicalmolecule, benzocaine H+, an analogue of PABAH+.27 Thismethod 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, thecollisional stripping does not aﬀect the protonation position.27
Here, we apply CAS-IR spectroscopy to PABAH+(H2O)n andexperimentally 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+(H2O)n(n = 0–7). After stripping the water ligands, PABAH+ forms van

Fig. 5 Calculated IR spectra of the (a) O- and (b) N-protomers of PABAH+(H2)4. (c) Observed CAS-IRPD spectra of PABAH+(H2O)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 comparedto the NH stretching modes.

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der Waals complexes with H2, which is introduced into thesecond ion trap together with the He buffer gas. Then, the IR
transitions of the PABAH+ monomer can be measured by loss of
H2 (tagging method36). Thus, all bands can be assigned byvibrational transitions of the monomer isomers. The spectrum
for n = 0 shows four bands in the 3400–3600 cm 1 range. Thesebands are in good agreement with the calculated spectrum ofO-protonated PABAH+ tagged by four H2 molecules (Fig. 5 andFig. S23, ESI†). The bands at 3435, 3496, 3535, and 3558 cm 1
are assigned to symmetric NH stretching, outward-facing OH
stretching, antisymmetric NH stretching, and OH stretching
toward the benzene ring, respectively. Only these bands areobserved 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 fourwater molecules.
In the CAS-IR spectrum of n = 5, new bands appear in therange from 3200 to 3350 cm 1. From the comparison to thetheoretical IR spectra shown in Fig. 5b, these bands at 3230,
3278, and 3303 cm 1 are assigned to symmetric and two
antisymmetric stretching vibrations of the NH3 group in the
N-protomer tagged with four H2 molecules. The theoretical
spectra also predict the OH stretching vibration at 3580 cm 1.
Probably this mode corresponds to the band at 3555 cm 1,
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 1 confirms the formation of the Nprotomer in the pentahydrated PABAH+ cluster, as well as theinterpretation of the IRPD spectra in the 6 mm range. Similarly,
the coexistence of O- and N-protomers in the PABAH+(H2O)nwith n = 6 and 7 is confirmed by the appearance of the bands inthe 3200–3350 cm 1 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 H2tagged PABAH+). The CAS-IR intensities are obtained by fitting
the CAS-IR spectra with Lorentzian profiles (details are shownin 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 kineticallytrapped and the N 2 O interconversion is not thermallyequilibrated. 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 protontransfer is more pronounced as the number of attached water
ligands is increased in the cluster. This observation suggeststhat the reaction barrier for the proton transfer is lowered bysequential addition of water molecules.

4. Summary
We have applied double ion trap IRPD and CAS-IR spectroscopy
to hydrated clusters of PABAH+. By selective formation of theThis journal is © the Owner Societies 2023

Paper
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 NH3+ and COOH groups areconnected by a H-bonded water chain.30 However, the bridged
conformer appears minor in the current IRPD spectrum for n =6 and the pentahydrated N-protomer of PABAH+ does not havesuch a bridged structure. Thus, we should consider a nonGrotthuss mechanism for the proton transfer, such as thevehicle mechanism, in this size regime.19,41,42 On the other
hand, the bridged structure becomes most abundant in higher
hydrated clusters (n = 7). This suggests that the Grotthuss
mechanism13,26,43,44 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 (D2O). 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 protontransfer reaction by raising the temperature of the reaction
trap. These future plans, as well as the application of double
ion trap spectroscopy31,32 to other molecules that contain two
or more protonation sites, will allow us to explore the mechanism of intracluster proton transfer in hydrated clusters andaqueous solution at the molecular level.

Conflicts of interest
There are no conflicts to declare.

Acknowledgements
This work was supported in part by KAKENHI (JP19K23624,
JP20K20446, JP20H00372, JP21H04674, and JP21K14585), theCore-to-Core program (JPJSCCA20210004) from Japan Society
for the Promotion of Science, research grant from World
Research Hub Initiative (WRHI) of Tokyo Institute of Technology, the Cooperative Research Program of the ‘‘Network Joint
Research Center for Materials and Devices’’ from the Ministryof Education, Culture, Sports, Science and Technology (MEXT),
Japan. The computations were performed at the ResearchCentre for Computational Science, Okazaki, Japan. M. F. is

Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 | 4487

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Paper
grateful for support from the Alexander von Humboldt foundation. O. D. acknowledges support from Deutsche Forschungsgemeinschaft (DFG, DO 729/10) and the World Research HubInitiative (WRHI) of Tokyo Institute of Technology.

Open Access Article. Published on 06 December 2022. Downloaded on 10/13/2024 6:48:37 PM.
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