An ionically tagged water-soluble artificial enzyme promotes the dephosphorylation reaction with nitroimidazole: Enhanced ionic liquid effect and mechanism

Article abstract of DOI:10.1021/acs.joc.5b00750

In this paper, we describe a novel synthesized ionically tagged water-soluble artificial enzyme (PI) that can efficiently cleave phosphate esters, with enhanced an ionic liquid effect through cooperative effects for the substrate activation and further nucleophilic reaction. The dephosphorylation reaction with PI was evaluated in the presence and absence of 2-methyl-4(5)-nitroimidazole, showing impressive rate enhancements of up to 2 × 10⁶-fold, ascribed to the imidazolide species known as excellent nucleophiles, and formed favorably at lower pH values in the presence of PI.

Full text of DOI:10.1021/acs.joc.5b00750

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Note
Ionically-Tagged Water Soluble Artificial Enzyme Promotes Dephosphorylation
Reaction with Nitroimidazole: Enhanced Ionic Liquid Effect and Mechanism
José G. L. Ferreira, Luciana Machado Ramos, Aline Lima de Oliveira, Elisa S. Orth, and Brenno A. D. Neto
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00750 • Publication Date (Web): 07 May 2015
Downloaded from http://pubs.acs.org on May 9, 2015
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Ionically-Tagged Water Soluble Artificial Enzyme Promotes
Dephosphorylation Reaction with Nitroimidazole: Enhanced
Ionic Liquid Effect and Mechanism
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José G. L. Ferreira, † Luciana M. Ramos,‡ Aline L. de Oliveira,‡ Elisa S. Orth*† and
Brenno A. D. Neto*‡
† Department of Chemistry, Universidade Federal do Paraná (UFPR), CP 19081, CEP
‡
81531-990, Curitiba, PR, Brazil.
Laboratory of Medicinal and Technological
Chemistry, University of Brasília, Chemistry Institute (IQ-UnB), Campus Universitário
Darcy Ribeiro, CEP 70904-970 – P.O.Box 4478 – Brasília, DF, Brazil.
Corresponding authors’ e-mail addresses: elisaorth@ufpr.br, brenno.ipi@gmail.com
Table of Contents/Abstract Graphic
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ABSTRACT
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In this paper, we describe a novel synthesized ionically-tagged water-soluble artificial
enzyme (PI) that can efficiently cleave phosphate esters, with enhanced ionic liquid
effect through cooperative effects for the substrate activation and further nucleophilic
reaction. Dephosphorylation reaction with PI was evaluated in the presence and absence
of 2-methyl-4(5)-nitroimidazole, showing impressive rate enhancements up to 2x106-
fold, accounted for by the imidazolide species known as excellent nucleophiles, and
formed favorably at lower pH values in the presence of PI.
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Key-words: artificial enzyme, nitroimidazole, dephosphorylation, ionic liquid, catalysis
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Dephosphorylation processes are a particular class of vital biological reactions,
intrinsically related to phosphorylated structures such as DNA and RNA. These
processes are indeed extremely unfavorable, therefore requiring enzymes.1 There is also
a great interest in developing novel bio-inspired catalytic systems that may help with
both the understanding about these enzymatic processes and the development of
artificial enzymes.2 Macromolecules are in this sense ideal backbones for this purpose
since they encompass active sites, as well as neighboring domains that can
synergistically assist the reaction. They can also assemble, leading to nanoreactors that
can concentrate reactants and thus accelerating the reactions. When designing new
catalytic systems, optimum reactive groups are required. In this context, much emphasis
is given to imidazole/imidazolium moieties that are present in many enzymatic active
sites because of their versatility. They may efficiently act as general acid-base pairs and
nucleophilic catalysts.3,4 The presence of imidazolium moieties also favors the so-called
ionic liquid effect with a typical enhancement in both yields and selectivities as a
consequence of ion-pairing and the formation of supramolecular aggregates.5 Although
the origin of the ionic liquid effect is hotly debated in the scientific literature, some
compelling evidences point to a cooperative cation-anion stabilizing effects.6 In
principle, ion pairs of imidazolium-based cations as neighboring polar domains could
assist the reaction more efficiently as a consequence of solubility effects and the ionic
liquid effect. Imidazolium-based derivatives are in this sense thought to be ideal due to
both their known high thermal and chemical stabilities (plus their nearly universal
solubility), which may certainly help to bring all reactants to the same phase, or to the
artificial synthetic enzyme domains, therefore facilitating the reaction to take place.
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Herein, we studied the dephosphorylation reaction promoted by a novel
synthesized ionically-tagged (imidazolium-based) water-soluble artificial enzyme (PI,
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Scheme 1), expected to act as a nanoreactor with enhanced ionic liquid effect. The
model substrate evaluated was the triester 2,4-dinitrophenyl phosphate (DEDNPP). The
incorporation of imidazolium cations should potentiate the ionic liquid effect in the
artificial enzyme toward dephosphorylation reactions and, to the best of our knowledge,
no ionically-tagged synthetic enzyme has been described before.
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Scheme 1. Synthesis of the water-soluble ionically-tagged artificial enzyme (PI).
We also studied the reaction of 2-methyl-4(5)-nitroimidazole (MNI) with
DEDNPP in the presence of PI in an aqueous medium, which is not possible to study
without PI since MNI is a water insoluble nucleophile. These nitrated imidazole
derivatives are of great interest since they constitute many pharmaceuticals7,8 and little
is known about their action, especially against phosphate esters, that could point to
possible carcinogenic activities. In fact, dephosphorylation processes are known to be
related to many dangerous diseases.
1
The PI structure was firstly characterized by H-15N HMBC experiments (see
Supporting Information, SI), typically used for the characterization of artificial enzyme
synthesized from commercially available polyethylene imines,9-11 and spectra show
typical hydrogens from the imidazolium-based ionic liquid structures (9.30 and 9.11
ppm for C2-H; 8-7 ppm C4-H and C5-H; 5.01 C7-H; 4.05 and 4.08 for methyl C6-H)
correlated with different types of nitrogen atoms at 167.0 ppm and 174.1 ppm (15N)
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from the imidazolium ring. As expected, the presence of varied signals for similar H
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15
and N atoms are the net result from the differences in chemical environments of the
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polymeric structure of PI. In addition, the signal at 25 ppm observed in the 15N analysis,
which is correlated with 1H at 2.80 ppm, reveals that tertiary amines are predominant in
the PI structure.
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Potentiometric titration were carried out to characterize PI as well as the system
PI/MNI, regarding the existing pKa´s. These provide important information in order to
infer the catalytic activity of specific species, since the pH effect in the reactions studied
(vide infra) will be evaluated. Fitting the titration profiles (see SI, Figure S2) with the
program BEST7, for PI, 4 values of pKa´s were determined: pKa1 =5.41, pKa2 =7.50,
pKa3 =8.99 and pKa4 =9.69 (error for all pKa´s 0.03), accounted to the amine sites
+
(NH2 ). Previous reports12 agree with the assumption of multiple pKa´s for a
macromolecule with several units of similar acid-basic moieties, attributed to the
neighboring groups that affect the equilibrium, e.g., stabilizing the deprotonated species.
The pKa of an amine group can therefore be shifted depending on whether it bears a
neutral or protonated amine group, just as observed in natural enzymes. The values are
additionally consistent with other complex amine-based macromolecules13 such as
lysozyme.14 In the case of MNI/PI, the same four pKa´s for PI were determined with an
additional pKa =10.48 0.01, attributed to MNI (formation of imidazolide species),
which agrees with other nitroimidazole derivatives.15
The reaction of DEDNPP with PI in the presence and absence of MNI was
followed at different pH; and the pH rate profiles obtained are shown in Figure 1 along
with data for the spontaneous reaction of DEDNPP in water.3 Results clearly show that
the dephosphorylation of DEDNPP is accelerated with PI and PI/MNI when compared
to the spontaneous reactions. It is also noted that kobs increases with pH for the reactions
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studied, suggesting that reactive species are formed at higher pH values. In the case of
solely PI, this can be attributed to the amine groups, potential nucleophilic sites, which
are knowingly reactive at higher pH, i.e., neutral. For PI/MNI, the imidazole group from
MNI can additionally act as nucleophilic sites, with a reactivity that depends on the pH,
evidencing the importance of the previous titration study. The data in Figure 1 were
fitted with Equation 1, which in all cases considers the reaction of DEDNPP with water
(k0) and hydroxide (kOH). With only PI, the last terms of the equation were not
considered, related to the MNI species. Although PI has many possible pKa´s for the
amine moieties (vide supra), only one pKa was required to fit the data, and two species
were relevant in this reaction: a partially protonated species (e.g. bipolar) with some
neutral amine groups with neighboring protonated groups (molar fraction χ PI+; kPI+),
and totally deprotonated species with neutral amine groups (χ PI-, kPI-). In the presence
of MNI, an additional species was considered, regarding the deprotonated imidazolide
group (χ MNI-, kMNI-), known to form at ca. pH 12 for nitroimidazoles.15 All these
possible pathways are illustrated in Scheme 2.
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PI
PI + MNI
H O
1E-3
1E-4
1E-5
2
7
8
9
10
pH
Figure 1. pH-rate profile for the reaction of DEDNPP with PI (3.14 mg/mL) in the
absence (ꢀ) and presence of MNI 0.02 M (●). Spontaneous hydrolysis of DEDNPP is
shown for comparison (ꢀ) at 25 °C.
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Scheme 2. Pathways for the possible reactions of DEDNPP with PI and PI/MNI.
kobs = k0 + kOH
[OH
]
+ kPI + χPI + + kPI − χPI − + kMNI χMNI
(1)
Table 1 presents the kinetic parameters obtained from the fitting data in Figure
1, where values of k0 and kOH are accordingly to previous studies.3 For the reaction of
DEDNPP with only PI, results show that the neutral species (PI-) is the most reactive, as
expected, leading to rate enhancements ~80-fold when compared to the spontaneous
reaction. Considering that PI has many of its nucleophilic sites hindered, acting more as
an aggregated ionic liquid, this reactivity is impressive. The kinetic pKa determined for
PI is consistent with the titration data, showing that above this critical value, the amine
moieties of PI are neutral, crucial for inferring nucleophilic reactivity. As mentioned,
macromolecules with multiple protonation sites with the same functionality (e.g. amine)
are known to have several pKas, since neighboring groups affect deprotonation of
subsequent sites.12 Multiple equilibria are however not crucial in the kinetic evaluated,
and an overall equilibrium can be reasonably considered (PI+ to PI-). The species PI+
has indeed little contribution but is necessary for fitting the data, evidencing that at
lower pH (<9.5) an overall partially neutral species of PI is reactive in the reaction
evaluated. The concentration of PI was varied, showing a linear profile with kobs (see SI,
Figure S3), characteristic of a nucleophilic attack. The obtained second-order constant
for PI was 0.11 g-1 mL-1 s-1, extremely high for macromolecule-mediated
dephosphorylation reactions. For example, the powerful α-nucleophilic
polyhydroxamate is effective in cleaving DEDNPP12 with kN= 0.017 g-1 mL-1 s-1,
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evidencing the high reactivity of PI. For the reaction with PI/MNI, similar constants for
PI were found and for MNI, rate enhancements were even more impressive: ~ 700-fold,
accounted for by the anionic imidazolide species (MNI-). It should be noticed that the
reaction with solely MNI cannot be evaluated since the substrate is water insoluble in
the absence of PI (even at low concentration, varying pH, with different surfactants,
etc.). Likewise, MNI is highly reactive at a considerably low concentration (0.02 M),
and the most commonly compared parameter in nucleophilic reactions is the second
order rate constant, which is 0.27 M-1 s-1 (among the highest reported with DEDNPP).
Even imidazole, that lacks the nitro group which readily decrease reactivity, presents
kN=0.177 M-1 s-1.3 The kinetic pKa determined for MNI is also consistent with the
titration study. Finally, the neutral species of MNI did not show significant reactivity.
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Table 1. Kinetic parameters obtained for the reactions of DEDNPP.
H2O, -OH: k0=8.0x10-6 s-1; kOH=0.42 M-1 s-1
Constant
kPI+ , s-1
kPI- , s-1
PI
1.88x10-5
5.97x10-4
-
PI / MNI
3.14x10-5
5.65x10-4
5.4x10-3
kMNI- , s-1
PI
9.69
pKa
MNI
pKa
-
10.56
Regarding the mechanism, the presence of imidazolium rings in the PI structure
is possibly helping in activating the substrate toward dephosphorylation reaction. A
remarkable acceleration on the transesterification reaction of some phosphate ester
derivatives in the presence of imidazolium-based ionic liquids has been demonstrated.16
Dupont17 and others18 have also proposed C=O activation in the presence of
imidazolium cations through the interaction of C=O---H-C2 between the acidic
hydrogen at C2 position of the imidazolium ring and the basic oxygen of the C=O
group. Based on the data obtained herein and on the literature evidences, an activation
mode could be proposed for DEDNPP (Scheme 3). In this sense, both parts of the
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synthetic enzyme play a role for the reaction acceleration. That is, the amine groups
(basic sites) and the imidazolium rings (acidic sites) are displaying a cooperative effect
for the dephosphorylation reaction.
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Scheme 3. Substrate activation by the imidazolium cation of PI.
Scheme 4 presents an overall mechanism proposed for the reactions studied
here. Thus, DEDNPP can be attracted to the PI neighboring domains by the polar
regions of the imidazolium cation, therefore being activated to react with the amine
sites. Nucleophilic attack by the amine sites occur via the following two paths: (A) on
the phosphoryl group leading to a phosphorylated intermediate (1); and path (B) on the
aromatic carbon affording intermediate 4. These intermediates are formed by a
concerted step3,19 and can readily decompose, regenerating PI and thus comprising a
catalytic nanoreactor with cooperative effects through the ionic liquid effect.
Analogous mechanisms of DEDNPP with amines have been reported3,19-23 but
without any ionic liquid effect exploited so far. In the present study, we propose the
attack on the phosphorus atom is predominant since the initial formation of the phenolic
product 2 should mostly come from path A, in accordance with the literature.19
Thermodynamic parameters were also obtained (Eyring plot given in the SI, Figure S4)
for the reaction of DEDNPP with PI, giving ꢀS‡ = -24.87 cal K-1 mol-1, ꢀH‡=15.5 kcal
mol-1, ꢀG‡ = 22.91 kcal mol-1 (25°C), which agree with the proposed nucleophilic
pathway.3
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In the case of the reaction of DEDNPP with PI/MNI, which should also be
attracted to the PI domains, an additional nucleophilic reaction should occur between
the anionic nitrogen of imidazolide (MNI) and the phosphoryl group, leading to the
intermediate 5, knowingly very unstable.3 Imidazolide is not usually studied since its
formation is associated with a very high pKa, over 14, for solely imidazole.15 Herein, the
withdrawing nitro group lowers this pKa (10.5, vide supra), enabling the formation of
the highly reactive species MNI-. This is also favored by the solubility and stabilizing
effects due to the presence of imidazolium-based neighboring domains in the synthetic
enzyme structure. The neutral species of MNI is not reactive, since the available
nitrogen is known to be a weak nucleophile (with a pKa < 3).15 Finally, an attack on the
aromatic carbon may be discarded, since previous studies showed that this path is
highly unfavorable with imidazole derivatives.3
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Scheme 4. Proposed mechanism for the studied reactions. Note the substrate activation
by the imidazolium cation has been omitted for clarity.
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Overall, results showed that PI may efficiently cleave phosphate esters, such as
DEDNPP, behaving as a catalytic nanoreactor with an enhanced ionic liquid effect
through cooperative effects for the substrate activation and further nucleophilic reaction.
PI proved to be capable of dissolving MNI in an aqueous medium, and this system
PI/MNI catalyzed the cleavage of DEDNPP with rate enhancements up to 2x106-fold,
comparing second order constant kN with the spontaneous reaction. The high reactivity
of MNI is mostly accounted for by the imidazolide species, known to act as excellent
nucleophiles, and formed favorably at lower pH values in the presence of PI (i.e. milder
conditions), in contrast to other imidazole-based systems (pH>13). As previously
mentioned, nitroimidazoles comprise many pharmaceuticals and the present results
evidence that precautions are necessary, since these compounds are potential
nucleophiles that can attack our biological phosphate esters (e.g. DNA, RNA), causing
defects, for example, and leading to tumoral processes. Lastly, these results have great
potential in designing water-soluble artificial enzymes with enhanced ionic liquid effect
and also detoxifying agents, since organophosphorus compounds constitute many
chemical weapons and pesticides that need to be monitored and eliminated (i.e.,
detoxification).24
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EXPERIMENTAL SECTION
Materials. MNI was obtained commercially and DEDNPP was prepared as described
previously.3 PI synthesis: Commercially available polyethylene imine (branched, Mw
~800 by GPC, Mn ~600 by GPC) was dried by azeotropic water removal. The
anhydrous PEI (5.00 g) was dissolved in anhydrous CH2Cl2 (10 mL) in a sealed Schlenk
tube. 1.40 g of 1-(2-chloroethyl)-3-methylimidazolium chloride was added followed by
2,6-lutidine (10 g). The mixture was heated at 100 °C for 2 h. After this time period, the
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Schlenk was unsealed and the mixture washed with CH2Cl2 followed by AcOEt
affording PI in nearly quantitative yield.
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NMR. All NMR measurements were carried out on a spectrometer (11.75 T) operating
1
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1
at 500.13 MHz for H and at 50.68 MHz for N. Typically, 2D H-15N HMBC pulse
sequence from the Bruker User Library was used. All experiments were performed
using 40 mg of PI in 600 µL of CD3OD in a NMR tube containing a sealed capillary
tube charged with nitromethane used as the external reference to set the scale (4.80 ppm
for 1H and 381.7 ppm for 15N).25
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Potentiometric titration. Potentiometric titration was carried out in a 100.0 mL
thermostated cell at 25°C. The solutions were acidified with HCl (0.11 M) and titrated
with small increments of KOH (9.95x10-2 M, CO2 free). The pH was monitored by a
pHmeter. The program BEST7 was used to determine the equilibrium constants.26
Kinetics. Reactions that were followed spectrophotometrically were started by adding
10.0 ꢁL from a stock solution of the substrate DEDNPP (7.5x10-3 M in acetonitrile) in
3.0 mL of the reaction solution, under pseudo-first-order kinetics. Solutions were
buffered with 0.01 M of KHCO3 (pH 7.0) and K2HPO4 (7.5-10.0). Reactions were
followed by the appearance of 2,4-dinitrophenol (2) at 400 nm with a thermostated cell
holder maintained at 25 ºC. Observed first-order rate constants (kobs) were calculated
from non-linear plots against time by using the Levenberg algorithm using the program
Origin8.
SUPPORTING INFORMATION
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NMR spectra, potentiometric titration curves and kinetic data. This material is available
free of charge via the Internet at http://pubs.acs.org
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ACKNOWLEDGMENTS
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The authors acknowledge the partial financial support from CNPq, CAPES, INCT-
Catalysis, INCT-Transcend group, FAPDF, DPP-UnB, Fundação Araucária, UFPR.
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