Supramolecular kinetic effects by pillararenes: The synergism between spatiotemporal and preorganization concepts in decarboxylation reactions

Article abstract of DOI:10.1039/d1nj00551k

A study about spontaneous decarboxylation reactions of 3-carboxy-1,2-benzisoxazole (CBI) nitrated derivatives (6-NitroCBI and 5,6-DinitroCBI) compared with supramolecular kinetic effects promoted by two cationic pillararenes (P5A and P6A) has been carried

Full text of DOI:10.1039/d1nj00551k

NJC
View Article Online
View Journal | View Issue
PAPER
Supramolecular kinetic effects by pillararenes:
the synergism between spatiotemporal and
preorganization concepts in decarboxylation
Cite this: New J. Chem., 2021,
45, 6486
reactions†
b
Eduardo Vieira Silveira, *a Rodrigo Montecinos,
Leandro Scorsin,a
Luis Garcia-Rio, *c Michelle Medeiros,a Vanessa Nascimento,
Faruk Nome,‡a
d
a
Ricardo F. Affeldt
and Gustavo A. Mickea
A study about spontaneous decarboxylation reactions of 3-carboxy-1,2-benzisoxazole (CBI) nitrated
derivatives (6-NitroCBI and 5,6-DinitroCBI) compared with supramolecular kinetic effects promoted by
two cationic pillararenes (P5A and P6A) has been carried out and brings contributions to advance
current theories related to supramolecular and enzymatic catalysis/inhibition. A higher energy barrier to
spontaneous hydrolysis of 6-NitroCBI relative to that of 5,6-DinitroCBI (DDG‡ = 2.98 kcal molꢀ1) was
determined experimentally, however similar transitions states were observed for both substrates. The
host–guest complexes generated catalytic effects up to 7.1-fold on kobs, except for 6-NitroCBICP5A
which is the only system that presented unprecedent inhibitory kinetic effects. The data (kinetics,
1H NMR titration and molecular dynamic simulations) elucidated the kinetic effects of the four
complexes as a result of their geometries, more specifically, by modifying 3 main factors: (1) number of
hydrogen bonds between solvent and carboxyl groups of the CBIs, (2) polarization of the O–N bond of
the isoxazole ring and (3) reduction of the inductive effect of NO2 groups by field effects. The data from
the present study encourage the use of both spatiotemporal and preorganization theories during inter-
pretations related to supramolecular and enzymatic catalysis.
Received 2nd February 2021,
Accepted 17th March 2021
DOI: 10.1039/d1nj00551k
rsc.li/njc
experimentally determinable and controllable. By the other side,3
the effects in preorganization theory can be briefly described by
1. Introduction
Research groups of different areas and distinct ideologies the large free-energy penalty already paid by preorganized dipoles
search for the elucidation of enzymatic catalytic effects. How- (commonly represented theoretically by water molecules) at
ever, these developed studies provided a discussion between two the enzymatic active site. Despite studies of vibrational Stark
very well-founded theories committed to solving these funda- effect spectroscopy already carried out on enzymes,4–6 the lack
mental questions, both always presenting valid arguments and of technology to investigate these fundamental issues can be
evidences. On the one hand,1,2 spatiotemporal theory postulates supplied by supramolecular chemistry using smaller and simpler
that such effects occur from an ideal proximity (B3 Å) and mimetic structures.
rigidity (or length time) between reactive groups, factors that are
Supramolecular chemistry shows high potential to solve
fundamental and practical issues related to different areas.
The remarkable characteristics of self-assembly control and genera-
tion of particular chemical environments with high affinity for
different classes of substrates offer fundamental conditions to
investigate and mimic natural biological structures.7,8 These char-
acteristics are observed in several supramolecular systems such as
by the use of different macrocycles to host–guest (H:G) complexa-
tion. For this purpose, pillararenes represent a recent class of
macrocycles with versatility already proven by the diverse studies
related to catalysis,9,10 analytical,11,12 environmental,13 food,14,15
materials,16,17 therapeutic/medical18,19 and among others. Further-
more, some studies suggest that the investigation of pillararenes
a
´
Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC,
88040-900, Brazil. E-mail: eduardovieira.chem@gmail.com
b
c
´
´
˜
Faculdad de Quımica, Pontificia Universidad Catolica de Chile, Av. Vicuna
Mackenna 4860, Santiago, Chile
´
´
´
´
´
Departamento de Quımica Fısica, Centro de Investigacion en Quımica Bioloxica e
Materiais Moleculares (CIQUS), Universidade de Santiago de Compostela,
15782 Santiago de Compostela, Spain
d
´
Department of Organic Chemistry, Fluminense Federal University, Niteroi, RJ,
24020-150, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1nj00551k
‡ Dr. Faruk Nome passed away on September 24, 2018.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
6486
| New J. Chem., 2021, 45, 6486–6494

View Article Online
Paper
NJC
may assist in the understanding of larger biological systems and 2. Experimental
their natural processes, for example, the catalytic effects promoted
2.1 Materials
by enzymes.9,10 This idea can be experimentally carried out in
The CBI derivatives and pillararenes were prepared on the basis
of the literature,28–45 with the modifications detailed in the
ESI.†
different ways including the use of sensitive kinetic probes such
as the 3-carboxy-1,2-benzisoxazole (CBI) and its derivatives.20
The 3-substituted-1,2-benzisoxazoles have been described as
a promising class of heterocycle with significant biological
activities,21–25 commonly associated with serotonergic and dopa-
minergic receptors.26 Belonging to this class, CBI is an organic
acid that has its decarboxylation reaction well studied in aqueous
and organic media,20,27–29 in the presence of surfactants/premi-
celles,30 antibody and albumin,31 cationic polymer,32 micelles,33
vesicles34 and quaternary ammonium macrocycles.35 Some parti-
cularities are notable for this reaction like the high sensitivity to
its ionization state (favored as carboxylate), polarity of the
medium (favored by polar solvents) and number of hydrogen
2.2 Kinetics
Reactions were followed with an UV-Vis spectrophotometer
equipped with a Peltier temperature controller set at 25.0 1C,
except to obtain the thermodynamic parameters, in which case
the temperature was varied between 25–65 1C. Reactions were
started by adding 20.0 mL of a CBI solution at 0.01 mol Lꢀ1
(in acetonitrile) to 2.0 mL of buffer solution in quartz cuvettes,
in the presence or absence of the pillararenes. All kobs were
measured by the appearance of the 5,6-DinitroCP or 6-NitroCP
at 380 and 400 nm, respectively. Except for the pH-rate profile,
the reactions were performed at pH 7.0 ([Bis–Tris methane] =
0.01 mol Lꢀ1).
bonds (HBs) between its COOꢀ group and solvent (favored by
28
aprotic solvents), reaching up to 108-fold increases on kobs
.
This high kinetic sensitivity allows us to propose the use of CBI
and its derivatives as potential probes to investigate the proper-
ties of chemical environments offered by hosts by monitoring
changes in kobs. Through theses studies important information
may potentially be able to collaborate with the spatiotemporal vs.
preorganization discussion.
2.3 ESI-MS
CBIs were solubilized in acetonitrile (B10 mM) and decarboxyla-
tion induced by sonication and heating, the expected m/z values
being confirmed for both CBIs. The samples were injected
directly to a mass spectrometry system consisting of a hybrid
triple quadrupole/linear ion trap mass spectrometer Q trap 3200
(Applied Biosystems/MDS Sciex, Concord, Canada). The experi-
ments were performed using the Turbo Ion SprayTM source
(electrospray ionization-ESI, Applied Biosystems/MDS Sciex,
Concord, Canada) in negative ion mode. Samples were infused
continuously at 10 mL minꢀ1 with a syringe pump. The capillary
needle voltage was maintained at ꢀ4.5 kV. The MS/MS parameters
were curtain gas, 18 psi; ion spray interface, 400 1C; gas 1 : 45 psi;
gas 2 : 45 psi; and collision gas, medium.
Here we describe an experimental and theoretical study about
spontaneous decarboxylation reactions of 6-NitroCBI (6-nitro-1,2-
benzisoxazole-3-carboxylic acid, Chart 1) and 5,6-DinitroCBI (5,6-
dinitro-1,2-benzisoxazole-3-carboxylic acid, Chart 1) compared
with supramolecular kinetic effects promoted by two cationic
pillar[n]arenes (n = 5 or 6) functionalized with trimethylammonium
groups (P5A and P6A, Chart 1). The experiments in pure water
(buffered when described) and in the presence of pillararenes
provided relevant information about CBI derivatives both indivi-
1
dually and when in complexes. Kinetics, H NMR titration and
molecular dynamics simulations are consistent with H:G stoi-
chiometry, the cavity size of macrocycles influencing the kinetic
effect type (catalysis or inhibition, unprecedented for the latter)
as well as the magnitude of binding (K1:1) and rate constants
(k1:1). The kinetic effects observed in these enzyme mimics could
not be explained with just one of the theories under discus-
sion (spatiotemporal or preorganization), but when both were
considered simultaneously.
2.4 NMR
For product characterization, CBIs were solubilized in acetone-
D6 and decarboxylation induced by sonication and heating.
The structures were confirmed by 1H and 13C{1H} analysis using
1
a Bruker AC 200 MHz spectrometer. For H NMR titrations, to
the NMR tube containing the CBI derivative were added solid
fractions of the pillararene and after total solubilization of each
addition the spectrum was collected.
2.5 Intrinsic reactions coordinates
Density Functional Theory (DFT) calculations were performed
at the M0646/6-31+G(d,p) level of theory using GAUSSIAN 09
package.47 The default parameters for convergence, viz. the
Berny analytical gradient optimization routine were used; conver-
gence on the density matrix was 10ꢀ9 atomic units, the threshold
value for maximum displacement was 0.0018 Å, and the maxi-
mum force 0.00045 hartree bohrꢀ1. All optimizations and frequency
calculations were performed using the Polarizable Continuum
Model (PCM) and the Solvation Model Density (SMD) of Truhlar
and coworkers.48 The transition states (TS, Fig. S11-b, ESI†) were
identified by their single imaginary frequencies.
Chart 1 Decarboxylation reactions of the 6-NitroCBI or 5,6-DinitroCBI in
the presence of the P5A or P6A to form the corresponding cyanophenolates
(CPs) and carbon dioxide.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 6486–6494
| 6487

View Article Online
NJC
Paper
2.6 Molecular dynamics simulations
firstly we studied the spontaneous decarboxylation reactions of
the 6-NitroCBI and 5,6-DinitroCBI at different acetonitrile : water
ratios. The results (Fig. S5 and Table S3, ESI†) showed that the
rate of reaction increases exponentially by reducing the fraction of
water, with increase in kobs of approximately 200 and 400-fold at
75% acetonitrile for 6-NitroCBI and 5,6-DinitroCBI, respectively.
These results corroborate the idea that nonpolar and protic
solvents stabilize the starting material and allowed us to later com-
pare the nanoenvironments offered by the pillararenes cavities
with acetonitrile percentage.
The kinetic pKa values (pKac) were also determined by data
fitting of kobs vs. H0/pH graphs (Fig. S6, ESI†), presenting values
of 1.61 and 1.18 for 6-NitroCBI and 5,6-DinitroCBI, respectively.
NMR and ESI-MS experiments confirmed the structure of the
cyanophenolates (CPs), the products of the decarboxylation
reactions (Table S1 and Fig. S2–S4, ESI†). Isotope effects (kw/kD,
Table 1) of 1.18 and 1.07 were observed to 6-NitroCBI and 5,6-
DinitroCBI, respectively. These subtle isotope effects can indi-
cate a destabilization of the TSs of the CBI derivatives during
decarboxylation reactions due to the lower vibrational frequency
of the O–D (oxygen–deuterium) bond. The smallest effect
observed for the 5,6-DinitroCBI could be associated with the
greater difference between its pKa and the corresponding of
water.36 The experimental activation parameters were obtained
for both derivatives from good Eyring relationship (Fig. S7, ESI†
Simulations were performed using the program package Gromacs
version 4.6.5.49 Pillararenes and CBI derivatives were built with
parameters from the GROMOS96 54A750 force field and solvated
with the SPC water model.51 Periodic boundary conditions were
applied in all three dimensions. All simulations were carried out
in the isothermal–isobaric ensemble. The temperature was main-
tained at 300 K using the Berendsen thermostat (v-rescale) with
pillararenes and CBI derivatives, and ions and water coupled
independently, with a coupling time constant of 0.1 ps. The
pressure was maintained at 1 bar using the Berendsen barostat
with a coupling time constant of 1.0 ps.52 The LINCS algorithm
was used to constrain the bond lengths of the pillararenes and
CBI derivatives and SETTLE to restrict the structure of the water
molecules. A 1.2 nm cut-off was used for the van der Waals
interactions. Long-range electrostatic interactions were calculated
using the particle mesh Ewald (PME).53 A time step of 2 fs was
used throughout the simulations. The neighbor list was updated
every 10 time steps. Both systems were equilibrated by 500 ps.
Trajectories of 20 ns were calculated for each system. To explore
the most probable structures formed by the interactions between
the compounds 6-NitroCBI and 5,6-DinitroCBI with P5A and P6A,
molecular dynamics simulations (MD) of the four systems were
performed. Each system containing one CBI derivative and one
macrocycle was solvated with 4714 water molecules (SPC) in the
presence of appropriate quantities of Brꢀ and Na+ to reach the
electroneutrality of the system.
and Table 1), with the favoring of 5,6-DinitroCBI (DDG‡
=
2.98 kcal molꢀ1) governed by the smaller enthalpy of activa-
tion, even with the half of the entropic gain when compared to
6-NitroCBI.
The computational study indicates the presence of TSs for
both substrates during the decarboxylation reactions. The IRCs
(Fig. S11-Above; calculated considering the optimized structure
3. Results and discussion
3.1 Spontaneous decarboxylation reactions
A study evaluated the influence of 24 solvents on the decarboxyla- of the reagent as the starting point, DE = 0, ESI†) represents a
tion reaction of the anionic 6-NitroCBI, showing that polar total energetic variation of DDE = 7.32 kcal molꢀ1 between both
solvents favor the reaction by modification of electronic effects, substrates as a result of a new electron withdrawing group
while aprotic solvents favor by modification of hydrogen bonding (NO2) added to 5-position in the benzisoxazole ring. The
(HBs, between solvent and its COOꢀ group).28 These kinetic molecular structures of the TSs (Fig. S11-Below, ESI†) assisted
effects represent increases up to 108-fold in kobs, making this by two water molecules corroborates the structures already
reaction an excellent probe to investigate the properties offered by reported for the CBI in aqueous medium,37–40 with the bonds
different chemical environments. Based on this particularity, cleaved (represented by the arrows) presenting similar distances
Table 1 Experimental and theoretical activation parameters of spontaneous decarboxylation reactions of anionic CBI derivatives. Dihedral angles and
interatomic distances for TSs of the same reactions (pH 7.00; [Bis–Tris methane] = 0.01 mol Lꢀ1; M06/6-31G+(d,p) theory level)
DG‡
DH‡
Distance
(kcal molꢀ1
)
CBI
Study
DS‡ (cal molꢀ1 Kꢀ1
)
Dihedral [C–C–C–O]
C–C (Å)
N–O (Å)
Experimental
Theoretical
24.98
24.77
32.42
25.16
24.95
1.31
—
90.511
—
1.76
—
2.15
Experimental
Theoretical
22.00
17.17
25.34
17.57
11.23
1.34
—
92.241
—
1.72
—
2.06
kW/kD(6-NitroCBI) = 3.00 ꢁ 10ꢀ6/2.55 ꢁ 10ꢀ6 = 1.18; kW/kD(5,6-DinitroCBI) = 4.65 ꢁ 10ꢀ4/4.34 ꢁ 10ꢀ4 = 1.07.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
6488
| New J. Chem., 2021, 45, 6486–6494

View Article Online
Paper
NJC
for both CBI derivatives. Another similar evidence for the two initially suggested a difference in the mode of accommodation
derivatives is related to the dihedral angle [C–C–C–O], which has inside the pillararenes cavities, that is, in the geometry of the
a torsion of approximately 901 in relation to the reagent. Corro- complexes. In the same way, the similarity between some binding
borating with the experimental data, the higher Gibbs free constants (or stabilization energies, DG) can be related to the
energy of activation (DG‡) calculated for 6-NitroCBI relative to formation of complexes of the same geometry. The similarity
5,6-DinitroCBI (DDG‡ = 7.60 kcal molꢀ1) is governed by the between the energy stabilization achieved by the complexes
enthalpy of activation, but here the activation entropy is similar 6-NitroCBICP6A (ꢀ22.85 kJ molꢀ1) and 5,6-DinitroCBICP6A
between the substrates. However, the highest calculated DDG‡ (ꢀ22.70 kJ molꢀ1) corroborates this idea, with P6A promoting
results from the divergence in enthalpy–entropy compensation up to 4.1 and 7.1-fold catalysis for the decarboxylation of
for 5,6-DinitroCBI, which can be a result of the lower enthalpic 6-NitroCBI and 5,6-DinitroCBI, respectively. In contrast, the
contribution from its late TSs structure. This latter affirmation is opposite kinetic effects and the discrepant energy stabilizations
in agreement with the smaller C–C atomic distances of its achieved by the complexes 6-NitroCBICP5A (ꢀ29.13 kJ molꢀ1
)
structure comparing with that already reported to CBI.37 These and 5,6-DinitroCBICP5A (ꢀ18.88 kJ molꢀ1) suggest different
information are presented in Table 1 together with their corres- geometric structures for these complexes. These factors were
ponding experimental data.
initially comprised by the smaller macrocycle cavity and the
additional NO2 group in the 5-position of the benzisoxazole ring
in the 5,6-DinitroCBICP5A system.
3.2 Kinetic effects of P5A and P6A
The catalytic effects were preliminarily understood by the
ability of the pillararenes to destabilize the CBI derivatives
based on reducing the number of HBs between water and its
COOꢀ groups. After observing the great proximity between the
charged groups of the complexes (COOꢀ and NMe3+) on
the molecular dynamics simulations data (MD, topic 3.4), the
possibility of COOꢀ groups to be found in the center of the
macrocycle cavity was disregarded. Thus, this reduction of HBs
was not attributed to the hydrophobicity in the pillararenes
cavities, but rather involves the high ability of NMe3+ groups to
modify the water structure. To demonstrate this, the decarboxyla-
tion reactions were investigated in the presence of different
tetramethylammonium bromide (TMA) concentrations in order
to simulate the high concentration of NMe3+ groups found in the
pillararenes portals. The influence of TMA on the structural
properties of water is widely studied,41–43 demonstrating that
cationic TMA+ presents low water concentration in its hydra-
tion layer, typical behavior of nonpolar compounds. The increase
of this effect occurs with its concentration, further reducing
the number of hydration water molecules and, consequently,
the number of HBs between them.
Fig. S8 (ESI†) shows exponential increments on kobs with
TMA concentration. This observation is not related to ionic
strength effects since several CBI derivatives have their reactivity
unchanged up to 1 mol Lꢀ1 of KCl.28 Therefore, the catalytic
effects observed for 6-NitroCBICP6A, 5,6-DinitroCBICP5A and
5,6-DinitroCBICP6A systems coincides with the spontaneous
decarboxylation rate constants of the corresponding derivative
at TMA concentrations of 2.02, 2.85 and 3.80 mol Lꢀ1, respec-
tively. These catalytic effects can also be correlated to the
chemical environments offered by the different acetonitrile
percentages, with the same sequence of the complexes corres-
ponding to the rate constants of the spontaneous reactions at
18.9, 17.1 and 23.4% acetonitrile. An exception to this idea
occurs in the 6-NitroCBICP5A complex, being its inhibitory
effect elucidated by NMR and MD data.
Substrates sensitive to different chemical environments (such
as CBI derivatives) are an alternative way to investigate the
properties offered by the pillararenes cavities observing the
generated supramolecular kinetic effects. Based on this idea,
the decarboxylation reaction of both anionic CBI derivatives
were studied at pH 7.00 in the presence of P5A and P6A. The
catalytic and inhibitory effects were initially expected for cavities
capable of destabilizing (as observed for polar and aprotic
solvents) and stabilizing (as observed for nonpolar and protic
solvents) the reagents, respectively. For the treatment of the
experimental kinetic data it was considered the simultaneous
decarboxylation reaction in water and in the pillararene cavity,
using eqn (S2) and (S3) (see ESI†) to evaluate the stoichiometry,
rate and binding constants (by non-linear fitting of the curves
kobs vs. pillararene concentration).
The kinetic effects of the pillararenes (Fig. 1 and Table 2)
showed modest increases on kobs with increasing pillararene
concentration, except for the 6-NitroCBICP5A system that
presented unprecedented inhibitory kinetic effect. The data
fitting (Fig. 1) were satisfactory for all four systems with H:G
model, this stoichiometry being confirmed for some systems by
1H NMR titration.
Even though both CBI derivatives belong to the same structural
class and exhibit similar sensitivity to the chemical environment,
the observation of opposite kinetic effects (catalysis and inhibition)
The experimental results for all systems are consistent
with H:G association, where decarboxylation reaction occurs
simultaneously in water and inside of the pillararene cavity.
Fig. 1 Influence of ( ) P5A and ( ) P6A concentration on the kobs for
decarboxylation reactions of (A) 6-NitroCBI and (B) 5,6-DinitroCBI ([CBIs] =
1.0 ꢁ 10ꢀ4 mol Lꢀ1; pH 7.00; [Bis–Tris methane] = 0.01 mol Lꢀ1; 25.0 1C).
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 6486–6494
| 6489

View Article Online
NJC
Paper
Table 2 Rate and binding constants of both anionic CBI derivatives in H2O, and P5A and P6A cavities (pH 7.00; 25.0 1C)
6-NitroCBI
5,6-DinitroCBI
Env.
k1:1 (sꢀ1
)
K1:1 (Mꢀ1
)
k1:1 (sꢀ1
)
K1:1 (Mꢀ1
)
H2O
P5A
P6A
(3.00 ꢂ 0.41) ꢁ 10ꢀ6
(7.77 ꢂ 0.53) ꢁ 10ꢀ7
(1.23 ꢂ 0.01) ꢁ 10ꢀ5
—
(4.65 ꢂ 0.10) ꢁ 10ꢀ4
(2.01 ꢂ 0.08) ꢁ 10ꢀ3
(3.30 ꢂ 0.05) ꢁ 10ꢀ3
—
(1.27 ꢂ 0.21) ꢁ 105 a
(2.03 ꢂ 0.21) ꢁ 103 c
(1.01 ꢂ 0.06) ꢁ 104 b
DG = ꢀ18.88 kJ molꢀ1
(9.49 ꢂ 0.70) ꢁ 103 d
DG = ꢀ29.13 kJ molꢀ1
.
DG = ꢀ22.85 kJ molꢀ1
.
.
DG = ꢀ22.70 kJ molꢀ1 (calculated by DG = ꢀRꢃTꢃln K1:1).
a
b
c
d
accommodation in the region of greater magnetic shielding in
the pillararenes cavities (DdP5A = 1.26 ppm; DdP6A = 0.24 ppm).
We emphasize that all hydrogens from pillararenes also dis-
played upfield shift when in the complex without reaching a
plateau, a continuous deshielding that indicates the association
of Brꢀ with increasing pillararene concentration.44 Another
important observation is the splitting of P5A hydrogens
(Fig. S9 and S10, ESI†) in the order of H2 c H5 D H3, a first
indication of the external accommodation of the 6-NitroCBI
where there is great magnetic influence on one of the P5A
portals (effects not observed for its complexation with P6A).
The successive spectra also provide an experimental evidence
regarding the 6-NitroCBICP5A inhibitory complex. The significant
shielding effect on the methylene bridge hydrogens (DdH4 D 0.16,
Fig. S10, ESI†) suggests the accommodation of the bulky NO2
group in the center of the P5A cavity, a condition that would
displace the high electronic density of the P5A cavity to the H4
hydrogens. These considerations suggest that kinetic inhibition
could occur partially by donor field effects (by P5A arenes) on the
NO2 group, which may directly reflect the reduction of its electron
withdrawing inductive effects. This is also supported by the MD
data along with additional factors involved in reaction inhibition.
The data fitting of Dd vs. pillararene concentration (Fig. 2,
eqn (S4), ESI†) provided K1:1 values of 4201 ꢂ 1513 Mꢀ1 and
429.68 ꢂ 69.76 Mꢀ1 for 6-NitroCBICP5A and 6-NitroCBICP6A,
respectively. Comparing with the kinetic data, this reduction in
K1:1 (30-fold with P5A and 23-fold with P6A) suggests the hydro-
phobic effects as one of the main driving forces involved in the
association of 6-NitroCBI with both pillararenes. In energetic
terms this is represented by the loss in energy stabilizations of
Scheme 1 General representation of the H:G association between a CBI
derivative and a P[n]A, with decarboxylation reaction occurring in both
environments (H2O and inside of the P[n]A cavity).
These considerations led us to Scheme 1, which represents the
generic behaviour involving kinetic and binding parameters for
the four studied systems.
3.3 1H NMR titration
Usually, hydrogen signals from host and guest when in the
complex present upfield or downfield shifts giving information
about the system, being the 1H NMR titration a powerful
alternative to elucidate the stoichiometry, affinity and the struc-
ture of the complexes. The fast decarboxylation of 5,6-DinitroCBI
and its catalysis by both pillararenes as well as by organic solvent
fractions compromised the accuracy in obtaining the succes-
sive spectra. Thereby, the NMR data correspond only to the
6-NitroCBICP5A and 6-NitroCBICP6A systems using 20% of
organic solvent (to solubilize the 6-NitroCBI), with the modifi-
cation in the experimental conditions (relative to the kinetics)
still providing relevant information about the complexes.
The successive spectra showed similar behaviours for 6-NitroCBI
aromatic hydrogens in the presence of both pillararenes (Fig. S9,
ESI†), with the upfield shift when in the complex indicating its
Fig. 2 1H NMR chemical shifts of the aromatic protons of 6-NitroCBI in
response to the increased (A, ) P5A and (B, ) P6A concentration, data
fitting as H:G model. The inset is the molar ratio method applied to the same
data ([6-NitroCBI]P5A = 12.93 mmol Lꢀ1; [6-NitroCBI]P6A = 7.65 mmol Lꢀ1
;
D2O : MeOD, 8 : 2, v/v; pD 7.00; 25.0 1C; 200 MHz).
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
6490
| New J. Chem., 2021, 45, 6486–6494

View Article Online
Paper
NJC
DDG = 8.45 and 7.82 kJ molꢀ1 for 6-NitroCBICP5A and similarities proven by the equivalence (P1, P2, P3, P4 and EL) or
6-NitroCBICP6A, respectively. These data further suggest that great similarity (P5) between parameters. Despite the same EL
in highly hydrophobic environments the system can act by contributions (ꢀ100 kJ molꢀ1), the VDW interactions were more
releasing 6-NitroCBI as a result of the energy destabilization of expressive for 5,6-DinitroCBI (ꢀ205 kJ molꢀ1) than 6-NitroCBI
the complexes, a perspective of great interest in the therapeutic/ (ꢀ165 kJ molꢀ1) due to the additional interactions promoted by
medicinal area mainly because several 3-substituted-1,2-benzi- the second NO2 group. The smallest number of HBs (P5) were
soxazole compounds are marketed as therapeutic drugs. The observed for these complexes, a parameter that confirms our
molar ratio method applied to Dd vs. [Pillararene]0/[6-NitroCBI]0 experimental results and proposal for the catalytic mechanism
(inserted in Fig. 2) has its inflection point in 1.0, what confirm promoted by the pillararenes, that is, reduction of HBs between
the H:G binding stoichiometry for the 6-NitroCBI complexation COOꢀ group and water.
with both pillararenes.
In contrast, the divergence among all parameters in the
complexes involving P5A can be broadly understood by its
smaller cavity size and by the additional NO2 group in one of
the CBI derivative, being the steric hindrance an unfavorable
and crescent phenomena in order of 6-NitroCBICP5A o 5,6-
DinitroCBICP5A. For the latter system, the additional NO2
group in the benzisoxazole ring results in its accommodation
0.10 nm more externally and 501 more distorted in the CPC
(compared to 6-NitroCBI), a condition that justifies its smallest
stabilization energy among the four systems (see Table 2).
The expressive fluctuation in P3 parameter indicates high
3.4 Molecular dynamics simulations
The most probable structures were investigated for the four
complexes, all presenting the nitrobenzene region of the CBI
derivatives inserted in the macrocycle cavity (Fig. 3). Accordingly
with our experimental data concerning stabilization energies
(Table 2), while the complexes formed with P5A presented
distinct geometries, P6A has the ability to accommodate both
CBIs equally. To explore these four structures as well as their
stability as a function of time five parameters (P1–P5) were
assessed: (P1) the average distance between carbon 6 (C6) from
CBI derivative and the center of the pillararene cavity (CPC); (P2)
the spatial orientation/angle of the CBI derivative in the CPC;
(P3) the average distance between charged groups (COOꢀ and
interaction dynamics between COOꢀ and NMe3 groups in
+
the complexes, which corroborates with both the smallest EL
and VDW energetic contributions as well as the largest number
of HBs with the solvent (P5). The highest P5 value observed for
6-NitroCBICP5A system was initially considered as a potential
factor involved in its inhibitory effects, but its similarity with P5
value for the 5,6-DinitroCBICP5A as well as the opposite
kinetic behaviour (catalysis and inhibition) observed for these
complexes suggested that more factors should be involved in
these effects.
+
NMe3 ); (P4) the energetic contributions resulting from electro-
static (EL) and van der Waals (VDW) interactions and (P5) the
number of HBs between water and COOꢀ group from CBI
derivative. All of these parameters are well discussed below
and some graphics presented in ESI† (Fig. S12–S16).
The five parameters calculated for all complexes (Table 3)
showed in detail some particularities of the systems and helped
in understanding both catalytic and inhibitory kinetic effects
promoted by the nanoenvironment offered by the pillararenes
cavities. The two complexes involving the P6A have their structural
In order to explore additional factors involved mainly in the
inhibitory kinetic effects the atomic charges (Table 4) and the
electrostatic potentials (ELPs, Fig. S17, ESI†) of the studied CBI
derivatives were calculated for all environments (H2O and in
the pillararenes cavities) using PCM method and the geometric
structure of the complexes. During the single step of the CBI
decarboxylation, the O–N bond cleavage (from isoxazole ring)
occurs with gain of electronic charge in the oxygen atom to
form the corresponding cyanophenolate. Based on this premise
and the calculated atomic charges the high polarization of the O–N
bond in the 6-NitroCBICP5A system proves to be an important
factor involved in the inhibitory kinetic effects. In other words, the
reduced ability of the oxygen atom to gain electronic charge in TS
makes the reaction kinetically unfavorable.
The donor field effects already mentioned in the ‘‘1H NMR
titration’’ topic (promoted by the high electronic density from
the P5A cavity on NO2 group in the inhibitory system) was
confirmed by the increasing in the electronic charges for both
oxygen and nitrogen atoms of the NO2 group. We also emphasize
that these electronic effects can present a direct dependence on
each other, for example, the high polarization of the O–N bond
(from isoxazole ring) being influenced by the reduction of the
electron withdrawing inductive effects of the NO2 group. The
considerations up to this point provide a broad perspective
regarding the studied systems, with the simultaneous participation
Fig. 3 Snapshots of the H:G complexes formed between CBI derivatives
and cationic pillararenes. (above) External interaction of 6-NitroCBI and
5,6-DinitroCBI with P5A. (below) Deeper insertion of the 6-NitroCBI and
5,6-DinitroCBI in the P6A cavity. Pillararenes and CBI derivatives are repre-
sented in thin sticks and VDW, respectively.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 6486–6494
| 6491

View Article Online
NJC
Paper
Table 3 Five parameters calculated for the four complexes formed between CBI nitrated derivatives and cationic pillararenes
P5A
P6A
Parameter
6-NitroCBI
5,6-DinitroCBI
6-NitroCBI
5,6-DinitroCBI
P1
P2
P3
P4
0.45 nm above
151 insertion
High fluctuation
ꢀ25 kJ molꢀ1 (EL)
ꢀ130 kJ molꢀ1 (VDW)
Average = 5
0.55 nm above
651 insertion
High fluctuation
ꢀ65 kJ molꢀ1 (EL)
ꢀ130 kJ molꢀ1 (VDW)
Average = 4
0.45 nm below
151 insertion
0.45 nm below
151 insertion
0.30 nm
0.31 nm
ꢀ100 kJ molꢀ1 (EL)
ꢀ165 kJ molꢀ1 (VDW)
Average = 2
ꢀ100 kJ molꢀ1 (EL)
ꢀ205 kJ molꢀ1 (VDW)
Average = 2
P5
Max. = 8
Max. = 7
Max. = 5
Max. = 4
P1, average distance between C6 and the CPC; P2, spatial orientation/angle of the CBI derivative in the CPC measured between two longitudinal
vectors (V1 = aligned to the CPC; V2 = formed by carbons C3 and C6 from CBI derivative); P3, average distance between charged groups (COOꢀ and
NMe3+); P4, energetic contributions from EL and VDW interactions; P5, number of HBs between water and COOꢀ groups from CBI derivative.
Table 4 Atomic charges of the oxygen and nitrogen atoms (from the isoxazole ring and NO2 groups) of the CBI nitrated derivatives in all environments
(H2O, P5A and P6A cavities)
H2O
P5A
P6A
Atom/group
6-NitroCBI
5,6-DinitroCBI
6-NitroCBI
5,6-DinitroCBI
6-NitroCBI
5,6-DinitroCBI
O-Ring
N-Ring
5-NO2
ꢀ0.447
ꢀ0.255
—
ꢀ0.452
ꢀ0.225
ꢀ0.551
ꢀ0.180
—
ꢀ0.436
ꢀ0.232
ꢀ0.462
ꢀ0.217
—
ꢀ0.436
ꢀ0.212
ꢀ0.379 (O)
ꢀ0.402 (O0)
0.364 (N)
ꢀ0.385 (O)
ꢀ0.389 (O0)
0.364 (N)
ꢀ0.354 (O)
ꢀ0.358 (O0)
0.207 (N)
ꢀ0.380 (O)
ꢀ0.405 (O0)
0.192 (N)
ꢀ0.371 (O)
ꢀ0.361 (O0)
0.178 (N)
ꢀ0.363 (O)
ꢀ0.371 (O0)
0.137 (N)
6-NO2
ꢀ0.420 (O)
ꢀ0.416 (O0)
0.383 (N)
ꢀ0.446 (O)
ꢀ0.452 (O0)
0.215 (N)
ꢀ0.409 (O)
ꢀ0.411 (O0)
0.067 (N)
of steric and electrostatic effects naturally extending to larger can be fundamentally better represented considering the spatio-
discussions related to supramolecular and enzyme catalysis/ temporal and preorganization theories, with the postulates of
inhibition.
both together providing greater deductive power than each one
separately.
4. Conclusions
Author contributions
In this work, experimental and theoretical studies about sponta-
neous hydrolysis and supramolecular kinetic effects by pillararenes
on decarboxylation reaction of two CBI nitrated derivatives pro-
vided conclusive information about the components (CBIs and
macrocycles) both individually and when in the complex. Despite
that both CBI derivatives present similar transition states during
spontaneous decarboxylation reaction, the additional NO2 group in
5,6-DinitroCBI resulted in a lower energy barrier during its decom-
position. In the presence of pillararenes both CBI derivatives
complexed in H:G stoichiometries, being the particularity of each
one in accommodating (geometry of complexes) the main deter-
mining factor between catalytic or inhibitory effects as well as the
magnitude of rate and binding constants. Specifically, intrinsic
effects involving the polarity modification of specific regions of
E. V. S. obtained and processed the kinetics and NMR data. The
theoretical study was performed by R. M. (molecular dynamics)
and L. S. (TSs and IRCs). V. N. performed the synthetic procedures.
The manuscript was written by E. V. S. This work was supervised
by L. G. R., M. M., F. N., R. F. A. and G. A. M. All authors have given
approval to the final version of the manuscript.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
the CBI derivatives, changes in the number of hydrogen bonds We dedicate this work on the memory of Professor Faruk Nome
(between solvent and COOꢀ group from CBI) and field effects on who passed away (September 24, 2018) during the preparation
NO2 groups were the main factors responsible for the observed of this paper. His professional and personal efforts have made
kinetic effects. Additionally, the effects observed on this model this possible. We thank Brazilian funding agencies CNPq,
´
reaction stimulate new work and applications of pillararenes CAPES and FAPESC, Central de Analises (UFSC) for NMR
as supramolecular catalysts, functional group protectors in chemical analysis and CEBIME (UFSC) for HRMS/ESI-TOF analysis. RFA
´
reactions, chemical sensors, drug delivery and others. Finally, we thanks CNPq/INCT-Catalise for financial support (grant
envision that supramolecular and enzymatic catalytic processes 444061/2018-5) and FAPESC TO (no. 2019TR0847). LGR thanks
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
6492
| New J. Chem., 2021, 45, 6486–6494

View Article Online
Paper
NJC
21 S. P. S. Chandra and A. C. Sharada, Int. J. Innovative Pharm.
Sci. Res., 2014, 5, 1006–1010.
´
Ministerio de Economıa y Competitividad of Spain (CTQ2017-
84354-P), Xunta de Galicia (GR 2007/085; IN607C 2016/03) and
22 Y. Masuda, Y. Utsui, Y. Shiraishi, T. Karasawa, K. Yoshida
and M. Shimizu, Epilepsia, 1979, 20, 623–633.
23 N. Ashwini, M. Garg, C. D. Mohan, J. E. Fuchs, S. Rangappa,
S. Anusha, T. R. Swaroop, K. S. Rakesh, D. Kanojia, V. Madan,
A. Bender, H. P. Koeffler, Basappa and K. S. Rangappa,
Bioorg. Med. Chem., 2015, 23, 6157–6165.
´
Centro singular de investigacion de Galicia accreditation (2016–
2019, ED431G/09) and the European Union (European Regional
Development Fund-ERDF). R. M. thanks Fondecyt (1171870).
V. N. thank FAPERJ (E-26/202.911/2019) for financial support.
24 G. Zheng, T. Chen, X. Peng and S. Long, J. Chem., 2015,
2015, 1–5.
25 R. A. Shastri and J. S. Varudkar, Indian J. Chem., Sect. B: Org.
Chem. Incl. Med. Chem., 2009, 48B, 1156–1160.
References
1 F. M. Menger and F. Nome, ACS Chem. Biol., 2019, 14,
1386–1392.
`
26 A. Nuhrich, M. Varache-Lembege, J. Vercauteren,
R. Dokhan, P. Renard and G. Devaux, Eur. J. Med. Chem.,
1996, 31, 957–964.
27 M. L. Casey, D. S. Kemp, K. G. Paul and D. D. Cox, J. Org.
Chem., 1973, 38, 2294–2301.
28 D. S. Kemp and K. G. Paul, J. Am. Chem. Soc., 1975, 97, 7305–7312.
29 D. S. Kemp, D. D. Cox and K. G. Paul, J. Am. Chem. Soc.,
1975, 97, 7312–7318.
2 B. S. Souza, J. R. Mora, E. H. Wanderlind, R. M. Clementin,
J. C. Gesser, H. D. Fiedler, F. Nome and F. M. Menger,
Angew. Chem., Int. Ed., 2017, 56, 5345–5348.
3 G. Jindal and A. Warshel, Proteins: Struct., Funct., Genet.,
2017, 85, 2157–2161.
4 S. H. Schneider and S. G. Boxer, J. Phys. Chem. B, 2016, 120,
9672–9684.
30 L. Brinchi, P. Di Profio, R. Germani, L. Goracci, G. Savelli,
N. D. Gillitt and C. A. Bunton, Langmuir, 2007, 23, 436–442.
31 Y. Hu, K. N. Houk, K. Kikuchi, K. Hotta and D. Hilvert, J. Am.
Chem. Soc., 2004, 126, 8197–8205.
5 H. Lehle, J. M. Kriegl, K. Nienhaus, P. Deng, S. Fengler and
G. U. Nienhaus, Biophys. J., 2005, 88, 1978–1990.
6 Y. Wu and S. G. Boxer, J. Am. Chem. Soc., 2016, 138,
11890–11895.
32 J. J. Lee and W. T. Ford, J. Org. Chem., 1993, 58, 4070–4077.
33 P. Di Profio, R. Germani, G. Savelli, G. Cerichelli, N. Spreti
and C. A. Bunton, J. Chem. Soc., Perkin Trans. 2, 1996,
1505–1509.
7 C. Bohne, Chem. Soc. Rev., 2014, 43, 4037–4050.
8 L. A. Marchetti, L. K. Kumawat, N. Mao, J. C. Stephens and
R. B. P. Elmes, Chem, 2019, 5, 1398–1485.
9 D. G. Liz, A. M. Manfredi, M. Medeiros, R. Montecinos,
´
´
34 M. V. Scarpa, P. S. Araujo, S. Schreier, A. Sesso, A. G. Oliveira
and H. Chaimovich, Langmuir, 2000, 16, 993–999.
35 F. P. Schmidtchen and W. Germany, J. Chem. Soc., Perkin
Trans. 2, 1986, 135–141.
36 P. K. Grzyska, P. G. Czyryca, J. Purcell and A. C. Hengge,
J. Am. Chem. Soc., 2003, 125, 13106–13111.
37 J. Gao, J. Am. Chem. Soc., 1995, 117, 8600–8607.
38 H. Zipse, G. Apaydin and K. N. Houk, J. Am. Chem. Soc.,
1995, 117, 8608–8617.
B. Gomez-Gonzalez, L. Garcia-Rio and F. Nome, Chem.
Commun., 2016, 52, 3167–3170.
10 E. H. Wanderlind, D. G. Liz, A. P. Gerola, R. F. Affeldt,
V. Nascimento, L. C. Bretanha, R. Montecinos, L. Garcia-
Rio, H. D. Fiedler and F. Nome, ACS Catal., 2018, 8,
3343–3347.
11 M. Bojtar, J. Kozma, Z. Szakacs, D. Hessz, M. Kubinyi and
I. Bitter, Sens. Actuators, B, 2017, 248, 305–310.
12 Y.-M. Zhang, J.-X. Su, Q. Li, W.-J. Qu, X. Zhu, Y.-L. Leng,
S.-F. Xin, H. Yao, Q. Lin and T.-B. Wei, Supramol. Chem.,
2016, 29, 411–416.
´
´
39 O. Acevedo and W. L. Jorgensen, J. Am. Chem. Soc., 2005,
127, 8829–8834.
40 O. Acevedo and W. L. Jorgensen, Acc. Chem. Res., 2010, 43,
142–151.
13 T. Zhou, N. Song, H. Yu and Y. W. Yang, Langmuir, 2015, 31,
1454–1461.
41 J. Turner, A. K. Soper and J. L. Finney, Mol. Phys., 1992, 77,
411–429.
14 M. M. Tian, D.-X. Chen, Y.-L. Sun, Y.-W. Yang and Q. Jia,
RSC Adv., 2013, 3, 22111–22119.
15 R. R. Kothur, B. A. Patel and P. J. Cragg, Chem. Commun., 42 E. J. Nilsson, V. Alfredsson, D. T. Bowron and K. J. Edler,
2017, 53, 9078–9080.
Phys. Chem. Chem. Phys., 2016, 18, 11193–11201.
16 S. Pan, M. Ni, B. Mu, Q. Li, X.-Y. Hu, C. Lin, D. Chen and 43 H. E. Wirth, J. Phys. Chem., 1967, 71, 2922–2929.
´ ´
44 B. Gomez, V. Francisco, F. Fernandez-Nieto, L. Garcia-Rio,
L. Wang, Adv. Funct. Mater., 2015, 25, 3571–3580.
17 I. Nierengarten, S. Guerra, M. Holler, J.-F. Nierengarten and
R. Deschenaux, Chem. Commun., 2012, 48, 8072–8074.
´
M. Martın-Pastor, M. R. Paleo and F. J. Sardina, Chem. – Eur.
J., 2014, 20, 12123–12132.
18 R. Joseph, A. Naugolny, M. Feldman, I. M. Herzog, 45 E. V. Silveira, V. Nascimento, E. H. Wanderlind, R. F. Affeldt,
M. Fridman and Y. Cohen, J. Am. Chem. Soc., 2016, 138,
754–757.
G. A. Micke, L. Garcia-Rio and F. Nome, J. Org. Chem., 2019,
84, 9684–9692.
19 D. Wu, Y. Li, J. Shen, Z. Tong, Q. Hu, L. Li and G. Yu, Chem. 46 Y. Zhao and D. G. Truhlar, Theor. Chem. Acc., 2008, 120,
Commun., 2018, 54, 8198–8201. 215–241.
20 J. Catalan, C. Dıaz and F. Garcıa-Blanco, J. Org. Chem., 2000, 47 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
65, 3409–3415. M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
´
´
´
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 6486–6494
| 6493

View Article Online
NJC
Paper
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, 48 A. V. Marenich, C. J. Cramer and D. G. Truhlar, J. Phys.
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, Chem. B, 2009, 113, 6378–6396.
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, 49 S. Pronk, S. Pall, R. Schulz, P. Larsson, P. Bjelkmar,
´
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J. A. Jr. Montgomery, J. E. Peralta,
F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin,
R. Apostolov, M. R. Shirts, J. C. Smith, P. M. Kasson,
D. Van Der Spoel, B. Hess and E. Lindahl, Bioinformatics,
2013, 29, 845–854.
V. N. Staroverov, R. Kobayashi, J. Normand, 50 N. Schmid, A. P. Eichenberger, A. Choutko, S. Riniker,
K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar,
J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene,
M. Winger, A. E. Mark and W. F. Van Gunsteren, Eur.
Biophys. J., 2011, 40, 843–856.
J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, 51 H. J. C. Berendsen, J. R. Grigera and T. P. Straatsma, J. Phys.
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, Chem., 1987, 91, 6269–6271.
R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, 52 H. J. C. Berendsen, J. P. M. Postma, W. F. Van Gunsteren,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador,
A. DiNola and J. R. Haak, J. Chem. Phys., 1984, 81,
¨
J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas,
3684–3690.
J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, 53 T. Darden, D. York and L. Pedersen, J. Chem. Phys., 1993, 98,
Gaussian, Gaussian Inc., Wallingford CT, 2009. 10089–10092.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
6494
| New J. Chem., 2021, 45, 6486–6494