PCCP
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
Volume 25
Number 6
14 February 2023
Pages 4363–5294
This journal is © the Owner Societies 2023 Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 | 4481
Cite this: Phys. Chem. Chem. Phys.,
2023, 25, 4481
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, *
de
Shun-ichi Ishiuchi *
acd
and Masaaki Fujii *
abd
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
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.
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 it
changes the chemical properties of the molecule, such as redox
behaviour, optical activity, and hydrophobicity.
1–4
In addition,
some molecules vary their preferred protonation site depend-
ing on the surrounding environment such as solvent or counter
ion.
5–29
Such protonation site-switching obviously has a huge
effect on the chemical reactivity and biological activity of the
molecule.
12,28
Para-aminobenzoic acid (PABA) is a benchmark molecule for
protonation site-switching.
8–11,13–18,20–22,29
PABA has two low-
energy 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.
14
Infrared (IR) spectroscopy and ion mobility mass spectro-
metry (IMS) have previously been used to identify the protona-
tion site in polyhydrated clusters of PABAH
+
. Hebert and Russel
a
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo
Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama226-8503,
b
School of Life Science and Technology, Tokyo Institute of Technology, 4259
Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8503, Japan
c
Department of Chemistry, School of Science, Tokyo Institute of Technology, 2-12-1
Ookayama, Meguro-ku, Tokyo 152-8550, Japan
d
International Research Frontiers Initiative, Tokyo Institute of Technology, 4259
Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.
E-mail: [email protected]n.de
e
Institut fu
¨r Optik und Atomare Physik, Technische Universita
¨t Berlin,
Hardenbergstrasse 36, 10623 Berlin, Germany
†Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d2cp04497h
Received 27th September 2022,
Accepted 3rd December 2022
DOI: 10.1039/d2cp04497h
rsc.li/pccp
PCCP
PAPER
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
View Journal
| View Issue
4482 | Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 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 domi-
nated 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
1
) to determine how many water molecules are
necessary to stabilize the N-protomer.
10
They 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)givefreeNHstretchingbandssimilartothemonomer,
while new vibrational bands appear with high intensity in the
spectrum of n=6(althoughsuchbandsarealreadyweaklypresent
also for no6). 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 pro-
tonation 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 hydratedclustersbycollisionswithN
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 estab-
lished 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 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
33–35
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 clus-
ters 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 N-
protomer).
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–H
stretching range, and thus the new IRPD spectra recorded in
the mid-IR (6 mm) range will give clear evidence for the proto-
mer 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.
2. Experimental and
computational methods
A methanol solution of PABA (Wako, 10
5
M) with 0.5% of
formic acid is electrosprayed and the fine droplets are
Fig. 1 Molecular structure of O- and N-protomers of PABAH
+
.
Paper PCCP
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
This journal is © the Owner Societies 2023 Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 | 4483
desolvated in a glass capillary heated to B80 1C. The generated
ions are introduced into the vacuum via an ion funnel. The ions
of interest are mass-selected by a quadrupole mass spectro-
meter (QMS) and transported by a tapered hexapole ion guide.
The ions are hydrated in an octupole ion trap (reaction trap
31,32
)
with water vapour introduced by a pulsed valve. The tempera-
ture of the reaction trap is maintained at 80 K. Hydrated ions
are mass-selected in the second QMS and introduced into the
cryogenic quadrupole ion trap (QIT
37
)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 H
2
is introduced into the QIT by a pulsed valve
for collisional cooling of hydrated ions down to B10 K, and
subsequently H
2
molecules are attached to the hydrated
PABAH
+
cluster (H
2
tagging method
36
). The cold hydrated ions,
PABAH
+
(H
2
O)
n
(H
2
)
m
, are irradiated with a tuneable OPO/OPA
IR laser. The PABAH
+
(H
2
O)
n
photofragments produced by the
dissociation of all H
2
molecules are monitored by a time-of-
flight mass spectrometer. H
2
is weakly bound so that the
structure of the hydrated PABAH
+
(H
2
O)
n
clusters are not
perturbed.
36
All IRPD spectra are measured by monitoring the
fragment ion yield generated by H
2
dissociation as a function of
laser wavenumber.
When CAS-IR spectra are measured, hydrated ions are
directed into the QIT with high kinetic energy (B16 eV) by
lowering the offset voltage at the QIT entrance electrode. The
ions are then collisionally dissociated to bare PABAH
+
, and H
2
molecules are attached to PABAH
+
. The H
2
-detached photofrag-
ments are detected by scanning the wavenumber of the IR laser.
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.
39
Preliminary 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.
10,27
Relative
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
1
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
6mm 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†).
3. Results and discussion
Fig. 2 shows the IRPD spectra of PABAH
+
(H
2
O)
nr7
recorded in
the 1300–1800 cm
1
range. Based on previous studies of bare
PABAH
+
,
20
the bands observed in the IRPD spectrum are
assigned to C(OH)
asym
2
at 1508 cm
1
, C–NH
aryl
2
at 1519 cm
1
,
C–C(OH)
aryl
2
at 1570–1600 cm
1
, and NH
bend
2
at 1658 cm
1
. The
Fig. 2 IRPD spectra of PABAH
+
(H
2
O)
n
with n= 0–7.
PCCP Paper
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
4484 | Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 This journal is © the Owner Societies 2023
bands characteristic of the N-protomer of the PABAH
+
mono-
mer, i.e., the CQO stretch (1790 cm
1
) and NH
3
umbrella
(1470 cm
1
) 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 O-
protomer (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
1
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 (NHO ionic H-bonds). On the other
hand, the CQO stretch band is relatively insensitive to hydra-
tion because water molecules form H-bonds mainly around the
positively charged NH
3+
centre. Thus, the absence and presence
of a band above 1700 cm
1
can be attributed to the O- and N-
protomer, respectively. This argument means that only O-
protomers 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
1
. This band at 1748 cm
1
is assigned to the CQO
stretch of the N-protomeric core, and thus strongly suggests
that the hydration by five water molecules makes the N-
protomer 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
1
(1726 and 1748 cm
1
, 1725 and 1746 cm
1
, respec-
tively). 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 struc-
tures 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. 3a). The bands observed between 1500 and 1650 cm
1
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
1
.
However, no clear band is observed in the range above
1700 cm
1
, suggesting that the population of the N-protomer
in PABAH
+
(H
2
O)
4
is (at most) small and thus below the
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 O-
protomers. 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 H
2
-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.
Paper PCCP
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
This journal is © the Owner Societies 2023 Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 | 4485
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 N-
protomer core is less stable than the most stable one with an O-
protomer core by 12.1 kJ mol
1
. It should be noted that the NH
3
umbrella band for n= 4 is blue-shifted by B90 cm
1
(fre-
quency: 1547 cm
1
, IR intensity: 114 km mol
1
) and overlaps
with bands of the O-protomer, making this vibration insensi-
tive for protomer assignment.
In contrast to nr4, the IRPD spectrum of PABAH
+
(H
2
O)
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 the
two most stable pentahydrated PABAH
+
clusters with an N-
protomer core, for which the CQO stretches are calculated at
1730 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 in
pentahydrated PABAH
+
. Bands around 1630 cm
1
are attribu-
ted 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
1
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 vibra-
tional bands are observed in the 1700–1800 cm
1
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 N-
protomeric 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
+
(H
2
O)
6
(Fig. 4a, b and Fig. S15, ESI†). The different
frequencies of the two CQO stretch modes arise from the
presence and absence of H-bonding to a water molecule (see
corresponding 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 red-
shifted from that of bare PABAH
+
(1781 cm
1
) probably due
to the change of electron density upon hydration of the NH
3+
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 O-
protomers. 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 H
2
-tagged
molecules.
45
The calculated intensity of the band at 1638 cm
1
of the O-protomer core is 686 km mol
1
.
PCCP Paper
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
4486 | Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 This journal is © the Owner Societies 2023
group. We name herein the conformers with and without H-
bonding 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
1
).
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
1
less stable than the most stable N-
protomer (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 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 O-
protomer may overcome the reaction barrier for O -N proton
transfer. The produced ‘‘hot’’ N-protomer may then be kineti-
cally trapped in the cold reaction trap (80 K). This means that
the kinetically trapped N-protomer reflects the structural infor-
mation 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
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 prototypical
molecule, benzocaine H
+
, an analogue of PABAH
+
.
27
This
method measures the populations of O- and N-protomers after
stripping all water molecules from the protonated solute mole-
cule and thus the conformational distributions in the hydrated
clusters must not necessary be determined. Significantly, the
collisional stripping does not affect the protonation position.
27
Here, we apply CAS-IR spectroscopy to PABAH
+
(H
2
O)
n
and
experimentally determine the relative abundance of O- and N-
protomers 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
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.
Paper PCCP
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
This journal is © the Owner Societies 2023 Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 | 4487
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
1
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
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 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
1
. From the comparison to the
theoretical 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 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
1
.
Probably this mode corresponds to the band at 3555 cm
1
,
which overlaps with the one of the OH stretching of the O-
protomer, because this band appears more strongly than pre-
dicted for the O-protomer. Thus, the appearance of the bands
between 3200 and 3350 cm
1
confirms the formation of the N-
protomer 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
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 H
2
-
tagged 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 2O 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.
4. 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 ambi-
guity 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 N-
protomer (n= 6) in which the NH
3+
and COOH groups are
connected 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 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.
19,41,42
On 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 trans-
fer, especially in larger hydrated clusters. However, a clear
conclusion that can be drawn here for the smaller clusters
(nr6) is that if the Grotthuss mechanism plays a role in the
proton transfer, it would first require the formation of energe-
tically 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 popula-
tion, our measurements are currently limited up to the hydra-
tion 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 mecha-
nism of intracluster proton transfer in hydrated clusters and
aqueous 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), the
Core-to-Core program (JPJSCCA20210004) from Japan Society
for the Promotion of Science, research grant from World
Research Hub Initiative (WRHI) of Tokyo Institute of Technol-
ogy, the Cooperative Research Program of the ‘‘Network Joint
Research Center for Materials and Devices’’ from the Ministry
of Education, Culture, Sports, Science and Technology (MEXT),
Japan. The computations were performed at the Research
Centre for Computational Science, Okazaki, Japan. M. F. is
PCCP Paper
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
4488 | Phys. Chem. Chem. Phys., 2023, 25, 4481–4488 This journal is © the Owner Societies 2023
grateful for support from the Alexander von Humboldt founda-
tion. O. D. acknowledges support from Deutsche Forschungs-
gemeinschaft (DFG, DO 729/10) and the World Research Hub
Initiative (WRHI) of Tokyo Institute of Technology.
References
1 N. Gupta and H. Linschitz, J. Am. Chem. Soc., 1997, 119,
6384–6391.
2 M. K. Nazeeruddin, S. M. Zakeeruddin, R. Humphry-Baker,
M. Jirousek, P. Liska, N. Vlachopoulos, V. Shklover,
C.-H. Fischer and M. Gra
¨tzel, Inorg. Chem., 1999, 38,
6298–6305.
3 C. H. Cheon and H. Yamamoto, J. Am. Chem. Soc., 2008, 130,
9246–9247.
4 J. T. Mohr, A. Y. Hong and B. M. Stoltz, Nat. Chem., 2009, 1,
359–369.
5 D. H. Aue, H. M. Webb and M. T. Bowers, J. Am. Chem. Soc.,
1976, 98, 311–317.
6 N. Solca
`and O. Dopfer, Angew. Chem., Int. Ed., 2003, 42,
1537–1540.
7N.Solca
`and O. Dopfer, J. Am. Chem. Soc., 2004, 126, 1716–1725.
8 Z. Tian and S. R. Kass, Angew. Chem., Int. Ed., 2009, 48,
1321–1323.
9 J. Schmidt, M. M. Meyer, I. Spector and S. R. Kass, J. Phys.
Chem. A, 2011, 115, 7625–7632.
10 T. M. Chang, J. S. Prell, E. R. Warrick and E. R. Williams,
J. Am. Chem. Soc., 2012, 134, 15805–15813.
11 J. L. Campbell, J. C. Y. Le Blanc and B. B. Schneider, Anal.
Chem., 2012, 84, 7857–7864.
12 S. Warnke, J. Seo, J. Boschmans, F. Sobott, J. H. Scrivens,
C. Bleiholder, M. T. Bowers, S. Gewinner, W. Scho
¨llkopf and
K. Pagel, et al.,J. Am. Chem. Soc., 2015, 137, 4236–4242.
13 J. L. Campbell, A. M.-C. Yang, L. R. Melo and W. S. Hopkins,
J. Am. Soc. Mass Spectrom., 2016, 27, 1277–1284.
14 J. Seo, S. Warnke, S. Gewinner, W. Scho
¨llkopf, M. T. Bowers,
K. Pagel and G. von Helden, Phys. Chem. Chem. Phys., 2016,
18, 25474–25482.
15 A. L. Patrick, A. P. Cismesia, L. F. Tesler and N. C. Polfer, Int.
J. Mass Spectrom., 2017, 418, 148–155.
16 E. Matthews and C. E. H. Dessent, Phys. Chem. Chem. Phys.,
2017, 19, 17434–17440.
17 H. Xia and A. B. Attygalle, J. Mass Spectrom., 2018, 53, 353–360.
18 R. Kumar and H. I. Kentta
¨maa, J. Am. Soc. Mass Spectrom.,
2020, 31, 2210–2217.
19 K. Ohshimo, S. Miyazaki, K. Hattori and F. Misaizu, Phys.
Chem. Chem. Phys., 2020, 22, 8164–8170.
20 T. Khuu, N. Yang and M. A. Johnson, Int. J. Mass Spectrom.,
2020, 457, 116427.
21 P. R. Batista, T. C. Penna, L. C. Ducati and T. C. Correra,
Phys. Chem. Chem. Phys., 2021, 23, 19659–19672.
22 M. Demireva and P. B. Armentrout, J. Phys. Chem. A, 2021,
125, 2849–2865.
23 M. McCullagh, S. Goscinny, M. Palmer and J. Ujma, Talanta,
2021, 234, 122604.
24 T. Uhlemann, G. Berden and J. Oomens, Eur. Phys. J. D,
2021, 75, 23.
25 N. Takeda, K. Hirata, K. Tsuruta, G. D. Santis, S. S. Xantheas,
S. Ishiuchi and M. Fujii, Phys. Chem. Chem. Phys., 2022, 24,
5786–5793.
26 B. Ucur, A. T. Maccarone, S. R. Ellis, S. J. Blanksby and
A. J. Trevitt, J. Am. Soc. Mass Spectrom., 2022, 33, 347–354.
27 K. Hirata, F. Haddad, O. Dopfer, S. Ishiuchi and M. Fujii,
Phys. Chem. Chem. Phys., 2022, 24, 5774–5779.
28 G. D. Santis, N. Takeda, K. Hirata, K. Tsuruta, S. Ishiuchi,
S. S. Xantheas and M. Fujii, J. Am. Chem. Soc., 2022, 144,
16698–16702.
29 T. Khuu, S. J. Stropoli, K. Greis, N. Yang and M. A. Johnson,
J. Chem. Phys., 2022, 157, 131102.
30 M. J. Hebert and D. H. Russell, J. Phys. Chem. B, 2020, 124,
2081–2087.
31 B. M. Marsh, J. M. Voss and E. Garand, J. Chem. Phys., 2015,
143, 204201.
32 E. Sato, K. Hirata, J. M. Lisy, S. Ishiuchi and M. Fujii, J. Phys.
Chem. Lett., 2021, 12, 1754–1758.
33 J. A. Stearns, S. Mercier, C. Seaiby, M. Guidi, O. V. Boyarkin
and T. R. Rizzo, J. Am. Chem. Soc., 2007, 129, 11814–11820.
34 E. Garand, M. Z. Kamrath, P. A. Jordan, A. B. Wolk,
C. M. Leavitt, A. B. McCoy, S. J. Miller and M. A. Johnson,
Science, 2012, 335, 694–698.
35 J. G. Redwine, Z. A. Davis, N. L. Burke, R. A. Oglesbee,
S. A. McLuckey and T. S. Zwier, Int. J. Mass Spectrom., 2013,
348, 9–14.
36 M. Z. Kamrath, E. Garand, P. A. Jordan, C. M. Leavitt,
A. B. Wolk, M. J. Van Stipdonk, S. J. Miller and
M. A. Johnson, J. Am. Chem. Soc., 2011, 133, 6440–6448.
37 S. Ishiuchi, H. Wako, D. Kato and M. Fujii, J. Mol. Spectrosc.,
2017, 332, 45–51.
38 W. L. Jorgensen and J. Tirado-Rives, J. Am. Chem. Soc., 1988,
110, 1657–1666.
39 F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp,
M. Lipton, C. Caufield, G. Chang, T. Hendrickson and
W. C. Still, J. Comput. Chem., 1990, 11, 440–467.
40 M. Frisch, G. Trucks, H. Schlegel, G. Scuseria, M. Robb,
J. Cheeseman, G. Scalmani, V. Barone, G. Petersson and
H. Nakatsuji, et al.,Gaussian 16, Gaussian, Inc., Walling-
ford, CT, 2016.
41 K.-D. Kreuer, A. Rabenau and W. Weppner, Angew. Chem.,
Int. Ed. Engl., 1982, 21, 208–209.
42 Y. Matsuda, A. Yamada, K. Hanaue, N. Mikami and A. Fujii,
Angew. Chem., Int. Ed., 2010, 49, 4898–4901.
43 N. Agmon, Chem. Phys. Lett., 1995, 244, 456–462.
44 M.-P. Gaigeot, A. Cimas, M. Seydou, J.-Y. Kim, S. Lee and
J.-P. Schermann, J. Am. Chem. Soc., 2010, 132, 18067–18077.
45 R. Otsuka, K. Hirata, Y. Sasaki, J. M. Lisy, S. Ishiuchi and
M. Fujii, ChemPhysChem, 2020, 21, 712–724.
Paper PCCP
Open Access Article. Published on 06 December 2022. Downloaded on 2/20/2023 12:47:58 PM.
This article is licensed under a
Creative Commons Attribution 3.0 Unported Licence.
View Article Online
