Inhibitory and Cooperative Effects Regulated by pH in Host-Guest Complexation between Cationic Pillar[5]arene and Reactive 2-Carboxyphthalanilic Acid

Article abstract of DOI:10.1021/acs.joc.9b01377

The study of host-guest complexation between reactive 2-carboxyphthalanilic acid (CPA) and two cationic pillararenes has been carried out. Host-guest complexation with significant kinetic effects was observed only with the smaller cavity size pillararene (P5A). Kinetics in the pH range 1.50-6.40, ESI-MS, ¹H NMR titration, and ROESY experiments were performed to characterize the complexes. High binding stoichiometry (H:G₂) was observed for all CPA protonation states. The system is pH-dependent, and inversion of cooperativity (negative to positive) occurs by increasing the dianionic CPA^2- concentration (allosteric behavior). Toward physiological pH, association constant K_1:1 does not change (10⁴ M^-1), and K_1:2 increased from 10² to 10⁴ M^-1, as well as the inhibitory effect increased up to 222-fold. NMR results elucidated the structure of the complex and allowed us to create a map of H-H interactions that describes well the diversity and number of interactions in the complex. ?

Full text of DOI:10.1021/acs.joc.9b01377

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Article
Inhibitory and Cooperative Effects Regulated by
pH in Host-Guest Complexation Between Cationic
Pillar[5]arene and Reactive 2-Carboxyphthalanilic Acid
Eduardo V. Silveira, Vanessa Nascimento, Eduardo H. Wanderlind, Ricardo
Ferreira Affeldt, Gustavo Amadeu Micke, Luis García#Río, and Faruk Nome
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01377 • Publication Date (Web): 08 Jul 2019
Downloaded from pubs.acs.org on July 9, 2019
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The Journal of Organic Chemistry
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Inhibitory and Cooperative Effects Regulated by pH in Host-
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Guest Complexation Between Cationic Pillar[5]arene and Re-
active 2-Carboxyphthalanilic Acid
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Eduardo V. Silveira,*,† Vanessa Nascimento,†† Eduardo H. Wanderlind,† Ricardo F. Affeldt,*,† Gustavo
A. Micke,† Luis Garcia-Rio, ††† and Faruk Nome†,
¶
†Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis – SC, 88040-900, Brazil.
††Departamento de Química Orgânica, Universidade Federal Fluminense, Niterói – RJ, 24020-150, Brazil.
†††Centro de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Física, Uni-
versidade de Santiago de Compostela, 15782, Santiago de Compostela, Spain.
*eduardoifg@hotmail.com; ricardo.affeldt@ufsc.br
KEYWORDS: Pillararene, host-guest, inhibition, allosteric, spatiotemporal, amide decomposition
ABSTRACT: The study of host-guest complexation between reactive 2-carboxyphthalanilic acid (CPA) and two cationic pil-
lararenes has been carried out. Host-guest complexation with significant kinetic effects were observed only with the smaller cavity
size pillararene (P5A). Kinetics in the pH-range 1.50-6.40, ESI-MS, 1H NMR titration and ROESY experiments were performed to
characterize the complexes. High binding stoichiometry (H:G2) was observed for all CPA protonation states. The system is pH-
dependent and inversion of cooperativity (negative to positive) occurs by increasing the dianionic CPA2- concentration (allosteric
behavior). Towards physiological pH, association constant K1:1 does not change (104 M-1) and K1:2 increased from 102 to 104 M-1, as
well as the inhibitory effect increase up to 222-fold. NMR results elucidated the structure of the complex and allowed us to create a
map of H-H interactions that describes well the diversity and number of interactions in the complex.
INTRODUCTION
chemotherapeutic drugs (such as doxorrubicin), potentiating
anticancer treatment.
Since 2008 and still considered a recent class of macrocy-
cles,1 the pillararenes are widely used in several areas,2–5 main-
ly in catalysis and in the development of chemical sensors.6–9
Its use in the inhibition of organic reactions is a less common
practice, such as found for other types of host,10–13 where the
aim is to reduce the rate of reaction or reactivity of some spe-
cies. As in catalytic systems, inhibitory systems are also able
to provide very important information about the components
(host and guest), alone or as complex, being the information
extracted by the simple relationship between environment and
reactivity.
Moravetz et. al. have demonstrated the intramolecular reac-
tivity of 2-carboxyphthalanic acid (CPA) as a bifunctional
intramolecular catalysis model (Scheme 1).16 A perspective of
this catalytic effect in terms of the spatiotemporal theory is
able to provide a better understanding of the high enzymatic
efficiency, such as in aspartic proteases (accelerations up to
1010-fold).17 Similarly, the enzymatic inhibition can be under-
stood in the same way, that is, reduction of the catalytic effi-
ciency by the steric restriction of its catalytic groups. These
considerations make the CPA reactivity an excellent model
reaction for this study.
Very important applications can rise from the inhibition of
organic reactions. For example, the action of acetylcholines-
terase on acetylcholine was inhibited by the host-guest associ-
ation of the neurotransmitter with an anionic pillar[5]arene, a
model system for the treatment of Alzheimer’s disease.14 In
another work,15 a cationic pillar[6]arene incorporated into
micelles proved to be efficient in targeting into cancer cells
(via folate receptors), forming a host-guest complex with
intracellular ATP (adenosine-5’-triphosphate) inhibiting its
hydrolysis. Suppression of this energy source resulted in the
blockade of the efflux pump and reduced the elimination of
Inhibition on CPA decomposition is mainly based on the re-
striction of its two carboxyl groups. The concepts of unfavora-
ble position (spatio) and length time (temporal) can also be
applied to different types of intramolecular reactions, such as
decomposition18 and cyclization19. This strategy is of great
interest when one seeks to keep intact the properties and func-
tionalities of a particular species in a reactive environment, a
concept that can be extended to several areas, such as synthe-
sis and drug delivery.20,21
1
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Scheme 1. Acid dissociation equilibria of CPA and its de-
composition, based on the literature.16
the neutral CPA (CPAH2), monoanionic (CPAH-) and dianion-
ic (CPA2-) species were calculated in water and in the P5A
cavity as a function of pH (Figure 1B), with the approximation
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P5A
P5A
between pKa1 and pKa2 responsible for the reduction of
4.2 % of the CPAH- species (represented by the red arrow).
At this point the experimental data suggest that: (1) the
complex is pH-dependent; (2) the inhibitory effect of the reac-
tion increases with pH and (3) the high electrostatic stabiliza-
tion on the anionic CPA should result in increasing association
constants in the order of CPAH2 < CPAH- < CPA2-.
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Herein, we investigate the host-guest complex between two
cationic pillararenes (P5A and P6A) and CPA (Chart 1). The
pillararene with smaller cavity size (P5A) proved to be a high-
ly selective host with inhibitory kinetic effects on CPA de-
composition. We performed a complete study about the influ-
ence of pH on the kinetic and equilibrium parameters of the
system. Through different techniques (UV-Vis, ESI-MS and
NMR) we determined rate and association constants, coopera-
tivity effects, stoichiometry and structural elucidation of the
1
complex. From the H NMR data (titration and ROESY) we
created a “map of H-H interactions” of the complex, making
clear the great diversity and number of interactions in the
system. The properties of the investigated system make it a
good model for development of pH- and environment-
dependent inhibitors.
Chart 1. Structures of hosts (P5A and P6A) and guest (2-
carboxyphthalanic acid, CPA).
Figure 1. (A) Influence of pH on the first-order rate constant for
CPA spontaneous hydrolysis (black data) and in the P5A cavity
(green data), at 25.0 ºC ([CPA] = 1.0 × 10-4 M; [P5A] = 4.0 × 10-4
M). (B) Relative percentages of the CPA species in H2O (black
solid lines) and in the P5A cavity (green dashed lines) as a
function of pH.
RESULTS AND DISCUSSION
Kinetics. After elucidating the decomposition products of
CPA (Scheme 1 and SI), we studied its decomposition in the
presence of P5A and P6A in the pH-range of 1.50–6.40. Insig-
nificant inhibitory kinetic effects (2.5-fold) occurred with
P6A, while P5A caused high inhibition of the reaction. Figure
1A shows the pH-rate profiles of the spontaneous hydrolysis
of CPA (black data) and in the presence of P5A (green data),
with clear effects of inhibition towards the physiological pH.
Errors were avoided by using concentrations of P5A suffice to
ensure complexation of all CPA.
The influence of the P5A concentration on the kobs for CPA2-
decomposition was directly determined by kinetic experiments
at pH 6.00 (Figure 2A and Table 1). The stoichiometric analy-
sis by fitting data (Equations S2-S3) revealed the typical be-
havior of a H:G2 system with a strong positive cooperativity (α
= 10.9, calculated by α = 4K1:2/K1:1), indicating the modifica-
tion of the P5A cavity after binding of the first CPA2-. In other
words, the CPA2- P5A offers a more favorable binding site
Ì
The data fitting in Figure 1A (Equation S1) provided the
than the free P5A, and the difference between the free energy
w
pKa values of CPA in both water (pKa1 and pKa2w) and in the
variations
P5A
P5A cavity (pKa1
and pKa2P5A), occurring reduction of al-
most 1 unit after complexation. This increase in the acidity of
the CPA is a result of the electrostatic stabilization promoted
by the NMe3+ groups on its carboxyl groups, reaching ΔpKa1
=
-0.62 and ΔpKa2 = -0.77. Therefore, the relative fractions of
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Figure 2. Influence of P5A concentration on the kobs for CPA decomposition at pHs (A) 6.00, (B) 4.25, (C) 3.45 and (D) 2.50 ([CPA] = 1.0
× 10-4 M; 25.0 ºC).
Table 1. Rate and equilibrium constants to CPA in P5A cavity at pHs 2.50, 3.45, 4.25 and 6.00.
K1:1
(104, M-1)
K1:2
(M-1)
KG
(M-2)
pH
k1:1 (s-1)
k1:2 (s-1)
2.50
3.45
4.25
6.00
(1.87 ± 0.1) × 10-4
(1.16 ± 3.5) × 10-5
(1.81 ± 7.1) × 10-5
(1.50 ± 8.3) × 10-7
(1.21 ± 0.1) × 10-4
(1.82 ± 2.3) × 10-5
(2.30 ± 11.1) × 10-6
(6.31 ± 15.4) × 10-8
(1.69 ± 0.4) × 104
(7.15 ± 1.1) × 104
(3.58 ± 0.8) × 104
(2.29 ± 0.5) × 104 (a)
(997 ± 478)
1.68 × 107
9.22 × 108
4.69 × 109
1.42 × 109
(1.29 ± 2.4) × 104
(1.31 ± 0.6) × 105
(6.22 ± 2.6) × 104 (b)
of the subsequent associations gives us a quantitative energy
analysis, such as ΔΔG = ΔG1:2 - ΔG1:1 = -2.48 kJ·mol-1 (see
Table 2). The obtained rate constants remained in the order of
k1:1D = 10-7 s-1 and k1:2D = 10-8 s-1, magnitudes compatible with
the data of Figure 1A.
For the other species (CPAH2 and CPAH-) the direct deter-
mination of its parameters was difficult due to the complexity
of the system considering the distribution of all species in both
environments (H2O and P5A) and possible changes of stoichi-
ometry. Therefore, we investigated the influence of P5A con-
centration on the kobs at pHs 2.50, 3.45 and 4.25 (Figures 2B-C
and Table 1) to evaluate the stoichiometry, cooperativity and
association constants to compare with values obtained by the
thermodynamic cycle (Scheme 2), as shown later. Thus, we
emphasize that the data at these pHs should not be attributed
to a single species or species distribution due to variation with
increasing P5A concentration. The maximum and minimum
fractions of each species were calculated for theses pHs in
both environments and are shown in Table S3.
The data fitting at pH 2.50 were satisfactory for both H:G
and H:G2 stoichiometries (Figure 2D). However, the predomi-
nance of CPAH2 in both environments (H2O = 85.5 % and
P5A = 52.5 %) and the association constants obtained for H:G
(1.43 × 104 M-1) and H:G2 global (KG = K1:1 × K1:2 = 1.68 × 107
M-2) indicate the system as H:G2. This conclusion was made
after we obtained the same magnitude of KG to CPAH2
through calculations involving the thermodynamic
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