Programmed enzyme-mimic hydrolysis of a choline carbonate by a metal-free 2-aminobenzimidazole-based cavitand

Article abstract of DOI:10.1021/ol403612e

The hydrolysis of a choline carbonate through a metal-free, enzyme-like mechanism has been achieved using a 2-aminobenzimidazole-based deep cavitand as catalyst. The supramolecular catalysis involves three steps: host-guest binding, carbamoylation and enzyme-like hydrolysis. Interestingly the rate-determining step proceeds through a programmed hydrolysis of carbamoylcholine-cavitand intermediate that may be driven by water molecules surrounding the benzimidazole walls of the cavity.

Full text of DOI:10.1021/ol403612e

Letter
pubs.acs.org/OrgLett
Programmed Enzyme-Mimic Hydrolysis of a Choline Carbonate by a
Metal-Free 2‑Aminobenzimidazole-Based Cavitand
Bartolome Soberats,† Elena Sanna,† Gabriel Martorell,‡ Carmen Rotger,† and Antoni Costa*,†
́
†Departament de Química, Universitat de les Illes Balears, Ctra. Valldemossa, km 7.5, 07122 Palma de Mallorca, Spain
‡Serveis Cientificotec
̀
nics, Universitat de les Illes Balears, Ctra. Valldemossa, km 7.5, 07122 Palma de Mallorca, Spain
S
* Supporting Information
ABSTRACT: The hydrolysis of a choline carbonate through a
metal-free, enzyme-like mechanism has been achieved using a
2-aminobenzimidazole-based deep cavitand as catalyst. The
supramolecular catalysis involves three steps: host−guest
binding, carbamoylation and enzyme-like hydrolysis. Interest-
ingly the rate-determining step proceeds through a pro-
grammed hydrolysis of carbamoylcholine-cavitand intermedi-
ate that may be driven by water molecules surrounding the
benzimidazole walls of the cavity.
Our intention here is to use a benzimidazole-based cavitand
he development of artificial enzymes has recently attracted
Ta lot of attention due to their applications in chemistry, as the catalyst for a stepwise hydrolysis of choline carbonates.
biology, and medicine.1 It is well-known that natural enzymes
The new extended resorcinarene 1, bearing (2-ABI) groups,
was designed to offer an appropriate environment to
accommodate the ammonium group of the choline derivative
inside the cavity owing to the stable vase conformation of the
cavitand. Concomitantly, the 2-ABI moieties and their
hydrogen bonded structural water molecules may promote
the hydrolysis of choline carbonates in the upper rim of the
supramolecular pocket (see abstract image).
Previously, the hydrolysis of PNPCC was achieved through
supramolecular catalysts based on cavitands or cyclodextrin
bearing Zn(II) as an active Lewis acid. In these complexes, the
metal center was key to modulating both the binding event and
the hydrolysis.6c,13 However, natural cholinesterases do not
include any metal ion in the active center.
bind their substrates through weak interactions and selectively
place the appropriate functional groups to achieve the catalysis.
This approach helps chemists to design supramolecular systems
that are able to simulate specific enzymatic roles. A number of
supramolecular catalysts based on polymers,2 cages,3 cavities,4
and macrocycles,5 among others, have demonstrated enzyme-
like activity in a wide variety of reactions. However, only a few
examples of supramolecular catalysis that mimic the enzymatic
mechanisms and kinetics have been reported.6 Herein, we
report the metal-free, enzyme-like hydrolysis of p-nitrophenyl-
choline carbonate (PNPCC) catalyzed by the supramolecular
cavitand 1 (Figure 1). This reaction involves three steps, i.e.
host−guest binding, covalent bonding, and a rate-determining
hydrolysis that proceeds in days. It is remarkable that the final
hydrolysis may proceed by the nucleophilic attack of the
hydrogen bonded water molecules located between the 2-
aminobenzimidazole (2-ABI) walls of the cavity.
Resorcin[4]arene cavitands having extended aromatic walls
have been shown to be effective receptors for improved binding
to charged and neutral small guests.7 Work done mainly by
Rebek et al. have demonstrated that deep cavitands decorated
with a variety of functional groups at the upper rim fulfill
specialized functions, such as encapsulation,8 catalysis,9 or as
nanocontainers designed to stabilize unstable intermediates.10
In particular, benzimidazole-based cavitands are very effective as
hosts for alkylammonium compounds11 and hydrocarbons12 of
suitable size and shape. The reason for the notable binding
capabilities of benzimidazole cavities lies in their stable vase
conformations under humid conditions due to the presence of
hydrogen bonded water molecules bridging two consecutive
benzimidazole walls that fix the vase conformation (Figure 1b).
In this work, we prepared a metal-free cavitand 1, which was
obtained in moderate yields by a stepwise synthetic procedure
1
(see the Supporting Information). The H NMR of 1 showed
the diagnostic methine proton as a sharp triplet at 5.52 ppm in
D2O/MeCN-d3, indicating the existence of 1 in the vase (C4v)
form (Figure 3a). The addition of choline (Cho) or
acetylcholine (ChoAc) to 1 induces their complexation. The
large upfield shifts observed for the N(CH3)3 and NCH2
protons and 1H−14N HSQC experiments prove that the
trimethylammonium groups of choline derivatives were located
deep inside the cavity.11a The affinity of 1 for choline
derivatives was assessed by integration of the resonances of
free and bound guests. Both complexes, namely 1·Cho+ and 1·
ChoAc+, afforded values well above 104 M−1 that preclude their
1
exact determination by H NMR titrations. These complexes
were also observed by MALDI-TOF mass spectrometry at m/z
Received: December 13, 2013
Published: January 13, 2014
© 2014 American Chemical Society
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Letter
Figure 2. (a) Ideal catalyzed hydrolysis of PNPCC. (b) Real-time
UV−vis kinetic conversion curves at 298 K of a solution of PNPCC
(0.1 mM) in MeCN/H2O (99:1 v/v) buffered with 10 mM EtN(i-Pr)2
and 5 mM CF3CO2H containing cavitand 1 at different molar ratios: 0,
0.1, 0.2, 0.5, and 1.0 with respect to PNPCC. Blue dashed line: time
course for 2-aminobenzimidazole (2-ABI) (0.1 mM). (b) Dependence
of the apparent first-order rate constant on cavitand concentration
(0.01−0.1 mM). The data were fitted to a straight line (R2 = 0.998),
with the second-order rate constant of hydrolysis defined by the slope
of the line.
Figure 1. (a) Chemical structures of 2-aminobenzimidazole-based
cavitand 1, Choline (Cho), Acetylcholine (ChoAc), and p-nitro-
phenylcholine carbonate (PNPCC) iodide salts. (b) Energy-minimized
(PM3) perspective side view of cavitand 1 (C4v) including four
hydrogen bonded water molecules on the upper rim.
Table 1. Kinetic Parameters for the Reaction of PNPCC (0.1
mM) in the Presence of Variable Amounts of Cavitand 1 or
2-ABI
entry
catalyst
[PNPCC]/[1]
kobs × 10−3 mn−1
kobs/kuncat
1220.56 and 1262.56 assigned to caviplexes 1·Cho+ and 1·
ChoAc+, respectively, thus confirming that cavitand 1 forms
tight complexes with choline derivatives (see the Supporting
Information).
1
2
3
4
5
6
none
−
0.7 0.2
10.0 2.1
17.6 5.4
38.4 13.5
81.2 24.3
21.5 6.4
−
1
0.1
0.2
0.5
1
14
25
55
116
30
1
1
Kinetic studies of PNPCC hydrolysis with 1 were performed
in acetonitrile−water mixtures by adaptation of reported
procedures.6c,14 Hydrolysis of PNPCC under basic conditions
generally affords choline, CO2, and p-nitrophenol (Figure 2a)
which can be easily monitored by UV spectroscopy. Reactions
of cavitand 1 with PNPCC were carried out in MeCN/H2O
(99:1 v/v) buffered with EtN(i- Pr)2/CF3CO2H at different
concentrations of 1, and the formation of the p-nitrophenolate
anion was followed at 405 nm by UV measurements. The
experimental kinetic curves obtained for different concen-
trations of cavitand are shown in Figure 2b. Pseudo-first-order
rate constants (kobs) evaluated from the initial slopes were in
line with previous reports.15 Table 1 shows that the amount of
cavitand has a marked effect on the initial rates of the reaction
(entries 1−5). The linear dependence of the pseudo-first-order
rate constants with the concentration of cavitand indicates its
participation in the reaction. The apparent second-order rate
constant was 79.3 0.2 M−1 mn−1. It is noteworthy that the
effectiveness of cavitand 1 is higher than that of 2-ABI alone
(entry 6). 2-ABI also catalyzes the reaction of PNPCC, though
less effectively, thus confirming the active role of the 2-
aminobenzimidazole units of 1 in the event.
1
2-ABI
−
the formation and subsequent hydrolysis of a 1-cholinecar-
bamate intermediate takes place under these conditions. The
time course of a solution of 1 (0.5 mM) and PNPCC (0.5 mM)
1
in unbuffered MeCN−D2O (95:5 v/v) was followed by H
NMR spectroscopy, and the representative results are shown in
Figure 3. To slow down the reaction between 1 and PNPCC
and to detect the formation of 1·PNPCC+, the spectrum at t =
10 min was registered at 278 K. Figure 3b shows the
characteristic upfield signal at −1.28 ppm corresponding to
the (+NMe3) of bound PNPCC deep inside of 1. The
coexisting methine protons of 1·PNPCC+ and unbound 1
were also observed at 5.63 and 5.53 ppm, respectively.
Formation of the 1·PNPCC+ complex was confirmed by
MALDI-TOF mass spectrometry, with m/z 1385.423 assigned
to [1·PNPCC]+. Thereafter, the extensive carbamoylation of 1
to give the N-benzimidazoylcarbamate 2 was evident, and it was
complete after 18 h (Figure 3c). 1H NMR spectra taken during
this period shows the progressive loss of the C4v symmetry
characteristic of 1 and 1·PNPCC. MALDI-TOF mass
spectrometry also revealed the majority presence of a peak at
It was expected that the catalytic hydrolysis of PNPCC
afforded Cho, CO2, and p-nitrophenol (Figure 2a). However,
1H NMR experiments ran on mixtures of 1 and PNPCC reveals
that the reaction presented here does not proceed through a
single reaction step. Instead, a stepwise mechanism involving
1
m/z 1246.502 corresponding to 2. The broadened H NMR
spectra and the absence of any significant signal at negative
shifts indicate that the trimethylammonium “knob” is outside
the aromatic pocket. Interestingly, the cavitand portion remains
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Scheme 1. Reaction of PNPCC with 2-ABI
contrast with the carbamoyl-cavitand intermediate 2 these
carbamates, obtained by direct condensation of 2-ABI and
PNPCC, do not hydrolyze even after extended periods at room
temperature.
These results suggest that hydrolysis of carbamate inter-
mediate 2 does not proceed by simple nucleophilic attack of the
water molecules in the solvent matrix. Hence, decarbamoyla-
tion of 2 to afford 1·Cho+ may be enabled by water molecules
located near the upper rim of the cavitand. Molecular modeling
of 2 reveals that the trimethylammonium group is outside the
cavity and that the carbamoyl group adopts a planar
conformation. In this conformation, the carbonyl group of
the carbamate is susceptible to hydrolysis by pseudo-intra-
molecular nucleophilic attack of water molecules lying in the
interstice between benzimidazole walls.
In conclusion, this new metal-free cavitand provides the
appropriate environment to promote the stepwise PNPCC
hydrolysis in an enzyme-like process. It is of special interest that
the hydrolysis of PNPCC takes place slowly due to the
combined effect of the supramolecular recognition event and
nucleophilic attack of water molecules confined at the outer rim
of the carbamoylated cavitand 2. This is a unique example of
quasi-irreversible catalyst inhibition found in cavitands.
1
Figure 3. Representative H NMR spectra (600 MHz) of selected
regions of a stoichiometric solution containing 1 (0.5 mM) and
PNPCC in MeCN-d3/D2O (95:5 v/v) recorded at different time
intervals. Colored resonances are diagnostic signals for free 1 (a, blue),
1·PNPCC+ caviplex (b, red), carbamoylated cavitand (c, khaki), and
bound choline 1·Cho+ (d, green). The spectrum of 1·Cho+, prepared
independently, is included for comparison (e, green).
always unchanged in vase form as indicated by a permanent
diagnostic signal at around 5.6 ppm. This intermediate slowly
hydrolyzes affording the final complex 1·Cho+ which recovers
the symmetric appearance expected for a cavitand−choline
complex (Figure 3d,e). It is relevant that the complete
decarbamoylation of 2 proceeds in a period of around 30
days at 298 K.
Remarkably, carbamoylation followed by slow decarbamoy-
lation is the mechanism of inhibition operating in certain
cholinergic drugs used for treatment of dementia, Parkinson’s,
and Alzheimer’s diseases.16 Rivastigmine is a paradigmatic
example of this behavior. Inhibition of acetylcholinesterases
(AcChE) with rivastigmine occurs by a mechanism that
includes (i) initial formation of a complex with AChE prior
to carbamoylation, (ii) stoichiometric carbamoylation of a
histidine residue in the catalytic triad of AChE, and (iii) very
slow (hours to days) decarbamoylation of the enzyme.16 Due to
the slow decarbamoylation process, rivastigmine is classified as
a “pseudoirreversible” inhibitor of AChE. These results closely
parallel those of PNPCC with cavitand 1.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and characterization data for all new
compounds. 1D, 2D NMR and mass spectra of complexes. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: antoni.costa@uib.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the Spanish Ministry of Economy and
Competitiveness (CTQ2011-27512/BQU and CONSOL-
IDER-INGENIO 2010 CSD2010-00065, FEDER funds) and
The reaction of PNPCC with 2-aminobenzimidazole (2-ABI)
and the subsequent hydrolysis were also investigated for
comparison (Scheme 1). Depending on the experimental
conditions, the addition of PNPCC to a solution of 2-ABI in
MeCN−D2O (95:5 v/v) gave the benzimidazole carbamates 3
and 4 (see the Supporting Information).17 However, in striking
the “Direccio
́
General de Recerca, Desenvolupament Tecno-
del Govern Balear” (CAIB, Project 23/2011,
logic i Innovacio
̀
́
FEDER funds) are gratefully acknowledged. E.S. and B.S. thank
CAIB and FSE for a predoctoral fellowship.
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Letter
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