Phosphate Diester Cleavage Research Articles

Hydrolysis of phosphate diester catalyzed by new mononuclear copper(II) complexes containing selenium ligands

AbstractThree new copper(II) mononuclear complexes containing different organoselenium groups (1–3) were synthesized and characterized by the following techniques: elemental analysis, IR and UV‐Vis spectroscopies, electrochemical and conductimetric analysis, and mass spectrometry. Three new complexes, with substituents made in the para position of the aromatic portion of the N,N‐bis(2‐(phenylselanyl)ethyl)amine ligand: p‐OCH3 (1), p‐CH3 (2), and p‐Cl (3), were synthetized to compare with the already published complex (4), with no substituents. The ligand coordinates to the copper(II) center in a tridentate way with Se, N, Se as donor atoms. The hydrolytic activity in phosphate diester cleavage of the complexes was investigated using 2,4‐BDNPP as substrate. The modifications in the ligand are reflected in the difference between the catalytic and activation parameters, where the kcat values follow the order: 4 > 2 > 3 > 1.

Synthesis, structure and properties of new triiron(III) complexes: Phosphodiester cleavage and antioxidant activity

This paper describes the synthesis, structural and spectroscopic characterizations of two new trinuclear iron(III) complexes: [Fe3(LS)(μ –OH)2(μ -OAc)2]ClO4 (CS) and [Fe3(LSe)(μ –OH)2(μ -OAc)2]ClO4 (CSe) with two new phenol-based ligands containing chalcogen atoms (S or Se) in pendant arms. Both iron complexes were characterized using the following techniques: X-ray crystallography, elemental analysis, IR, Mossbauer and UV–vis spectroscopies, conductimetric analysis and mass spectrometry. The crystal structures of the complexes reveal that their coordination center is formed by three linearly arranged iron atoms, joined by μ -hydroxo, μ -phenoxo and μ -acetato bridges. The hydrolytic activity in the phosphate diester cleavage of CSe and CS was investigated using 2,4-BDNPP as a substrate, and applying the Michaelis-Menten approach, giving the kinetic parameters (CSe/CS): kcat (s−1) = 3.81 × 10-3/2.32 × 10-3 and a catalytic efficiency of E = kcat/ KM, (L/mol s−1) = 1.83/3.18. The complexes and ligands also showed antioxidant activity.

Binuclear metal complexes with a novel hexadentate imidazole derivative for the cleavage of phosphate diesters and biomolecules: distinguishable mechanisms

Oxidative cleavage of phosphate diesters (HPNP, BNPP) is highly faster than the hydrolytic one by binuclear metal complexes with novel imidazole derivative, producing a non-lactone phosphate monoester due to the direct attack of free radicals.

Hydrolytic activity of a crown ether-lanthanum complex in the phosphate diester and DNA cleavage

Hydrolytic activity of a crown ether-lanthanum complex in the phosphate diester and DNA cleavage

Mechanistic Aspects of Phosphate Diester Cleavage Assisted by Imidazole. A Template Reaction for Obtaining Aryl Phosphoimidazoles.

Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of o-(2'-imidazolyl)naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a SN2(P)-type mechanism involving a pentacoordinated phosphorus TS, with apical positions occupied by the incoming imidazolyl nucleophile and the p-nitrophenolate leaving group. The P-O bond to the leaving group is about 50-60% broken in the transition state.

Phosphate Diester Cleavage Promoted by the Metallomicelles of Ce(III) Complexes of Aza-Crown Ether with Different Numbers of Nitrogen Atoms

The design of artificial hydrolase has attracted extensive attention due to their scientific significance and potential application in the field of gene medicine and molecular biology. This work reports the catalytic activation of two aza-crown ether Ce(III) complexes and their metallomicelles as artificial hydrolase in bis(4-nitrophenyl) phosphate ester (BNPP) hydrolysis. The chemical composition of two complexes was determined by the fluorescence spectra and the mole ratio method for electronic absorption spectra. The bonding effect of BNPP and solubilizing effect of the complexes were proved by a method of fluorescence spectroscopy. The catalytic activity of different catalytic systems in BNPP hydrolysis was measured with UV-vis spectrophotometric method. These catalytic systems showed high catalytic activity for promoting BNPP hydrolysis at the almost physiological conditions. BNPP hydrolysis rate in these catalytic system is about 107- to 109-fold faster than that of the BNPP spontaneous hydrolysis in aqueous solution at the same conditions. The metallomicelle systems exhibited higher catalytic activity compared with the complex solution systems in BNPP hydrolysis, and hexadecyltrimethyl ammonium bromide micelle provides a useful catalytic environment for reaction. The acid effect of the catalytic system is ascribed to the formation of metal-bound hydroxide serving as a better kind of nucleophile.

Enhancing Phosphate Diester Cleavage by a Zinc Complex through Controlling Nucleophile Coordination.

Metal-ion complexes are the most effective artificial catalysts capable of cleaving phosphate diesters under mild aqueous conditions. A central strategy for making these complexes highly reactive has been to use ligand-based alcohols that are coordinated to the ion, providing an ionised nucleophile under neutral conditions but at the expense of deactivating it. We have created a highly reactive Zn complex that is 350-fold more reactive than an alcohol analogue by preventing the nucleophile binding to the metal ion. This strategy successfully delivers the benefits of efficient nucleophile delivery without strongly deactivating the metal ion Lewis acidity nor the oxyanion nucleophilicity. Varying the leaving group reveals that the transition state of the reaction is much further advanced than the reaction with hydroxide.

Phosphate Diester Cleavage, DNA Interaction and Cytotoxic Activity of a Bimetallic Bis(1,4,7‐triazacyclononane) Zinc Complex

AbstractThe dinuclear zinc complex [Zn2{bcmp(‐H)}(μ‐Cl)](ClO4)2·H2O {bcmp = 2,6‐bis(1,4,7‐triazacyclonon‐1‐ylmethyl)‐4‐methylphenol} has been synthesized and structurally characterized. The DNA binding affinity was assessed by ethidium bromide fluorescence quenching experiments. The complex relaxes supercoiled pUC19 DNA into the nicked form at low micromolar concentration. Mechanistic studies were carried out using the DNA and RNA models bis(2,4‐dinitrophenyl) phosphate (BDNPP) and 2‐hydroxypropyl p‐nitrophenyl phosphate (HPNP). A detailed kinetic analysis suggested that the bridging OH group of the solution species [Zn2{bcmp(‐H)}(μ‐OH)]2+ acts as the nucleophile in the hydrolysis of BDNPP, while in the case of HPNP, the bridging OH group acts as a general base and seems to shift to a terminal position upon substrate coordination. Finally, the cytotoxicity profile of the dinuclear zinc(II) complex was assessed. The complex showed promising in vitro antitumour activity against pancreatic and lung cancers cell lines.

Quantum mechanical/effective fragment potential (QM/EFP) study of phosphate diester cleavage in aqueous solution

We report a study of the P–O bond cleavage of the anion of 2,4-dinitrophenyl ethyl phosphate (2,4-DNPEP), promoted by methylamine. The aminolysis reaction was investigated in the gas phase and an aqueous environment using the hybrid quantum mechanical/effective fragment potential (QM/EFP) approach. According to the B3LYP/6-31++G(d,p) results, in the gas phase the cleavage of the P–O bond of 2,4-DNPEP proceeds in one step through a concerted mechanism, with an activation free energy of 39kcal/mol. Including solvent effects at the B3LYP/6-31++G(d,p)/EFP level changes the reaction to a two-step associative mechanism. The first step corresponds to the formation of the P–NHMe bond and the second step to concerted proton transfer and P–O(2,4-dinitrophenolate) cleavage. In the absence of any assistance of water molecules to promote the proton transfer, the second step is rate determining with an activation free energy for the breakdown of the intermediate of 21.8kcal/mol at the MP2/cc-pVTZ level and a free energy of activation of 32.3kcal/mol for the overall reaction. However, the proton transfer with the assistance of a water molecule may occur without significant energy barrier. The results show how solvent can play an important role in favoring an associative mechanism for the aminolysis of phosphate diesters, through the formation and stabilization of a pentacoordinate phosphorane intermediate.

Steric effects on the catalytic activities of zinc(ii) complexes containing [12]aneN3 ligating units in the cleavage of the RNA and DNA model phosphates

A series of N-methylated mono- and di-[12]aneN(3) ligands () have been synthesized and characterized. The steric effects on the catalytic activities of their mononuclear and dinuclear zinc(ii) complexes in the cleavage of a RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP, ) and a DNA model methyl 4-nitrophenyl phosphates (MPNPP, 4) in methanol have been investigated at 25 °C. In the cleavage of phosphate catalyzed by the mononuclear complexes, derived from the N-methylation in the [12]aneN(3) backbone, the plots of k(obs)versus [Zn(II)] changed from an upward curvature to linearity with increasing level of methylation, indicating that N-methylations led to a reduction of dinuclear association that was responsible for the synergetic effect. Compared to the activities of the complex with non-methylated di-[12]aneN(3) ligand, those of the dinuclear zinc(ii) complex (Zn(2)-8), which has the two N-methyl groups, were reduced by two orders of magnitude as measured by the second-order rate constants and synergetic effect in the cleavage of both model compounds. For reactions catalyzed by the fully N-methylated dinuclear complex (Zn(2)-9), no synergetic effect was observed. Nevertheless, complex Zn(2)-8 still showed the remarkable catalytic efficiency, with rate accelerations of 10(9-10)-fold in the cleavage of each of the two phosphates relative to the background reactions, and the synergetic effects of up to 561 folds. pH jump experiments confirmed that the rate-limiting step in the cleavage of by Zn(2)-8 involved the binding process, while that in the reaction with 4 was the chemical cleavage of the P-O bond. Steric effects in the cleavage reactions were analyzed in detail and were compared with the electronic effect caused by oxy anion bridging group in the di-[12]aneN(3) ligand and also with the hydrophobic effect observed in other systems. The work has further confirmed that the combination of the cooperativity between two metal ions and a medium effect could result in excellent catalytic activities for the cleavage of phosphate diesters.

Efficient promotion of phosphate diester cleavage by a face-to-face cyclodextrin dimer without metal

An organic face-to-face cyclodextrin dimer promotes the cleavage of bis(4-nitrophenyl) phosphate efficiently in neutral pH without the addition of metal. Both of the phosphate diester bonds can be cleaved.

Phosphate diesters and DNA hydrolysis by dinuclear Zn(II) complexes featuring a disulfide bridge and H-bond donors

The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl)amine (BAPA) metal binding unit and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for phosphate diesters. The Zn(II) complexes of BAPA are known to elicit the cooperation between the metal ion and the hydrogen-bond donating amino groups to greatly increase the rate of cleavage of phosphate diesters. The reactivity of the dinuclear complex 1·Zn(II) 2 toward bis- p-nitrophenyl phosphate and plasmid DNA was investigated and compared with that of reference complexes devoid of the disulfide bridge or of the hydrogen-bond donating amino groups. The dimetallic Zn(II) complex produces remarkable accelerations of the rate of cleavage of both the substrates accompanied by significant differences. In the case of BNP, the presence of the disulfide bridge does not lead to the improvement of the cooperative action of the two metal ions expected as the result of better preorganization. On the other hand, in the case of DNA the complex 1·Zn(II) 2 is much more reactive that the corresponding reference devoid of the disulfide bridge. Hence, different requisites must be fulfilled by a good catalyst for the cleavage of the two substrates. Moreover, binding studies with DNA indicated that the presence of two metal ions in the complex or of the pyridine amino groups, but not of the disulfide bridge, results into an enhanced affinity of the complexes toward this substrate.

Phosphate Binding Energy and Catalysis by Small and Large Molecules

Catalysis is an important process in chemistry and enzymology. The rate acceleration for any catalyzed reaction is the difference between the activation barriers for the uncatalyzed (Delta G(HO)(#)) and catalyzed (Delta G(Me)(#)) reactions, which corresponds to the binding energy (Delta G(S)(#) = Delta G(Me)(#)-Delta G(HO)(#)) for transfer of the reaction transition state from solution to the catalyst. This transition state binding energy is a fundamental descriptor of catalyzed reactions, and its evaluation is necessary for an understanding of any and all catalytic processes. We have evaluated the transition state binding energies obtained from interactions between low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound substrate. Work on catalysis by small molecules is exemplified by studies on the mechanism of action of Zn2(1)(H2O). A binding energy of Delta G(S)(#) = -9.6 kcal/mol was determined for Zn2(1)(H2O)-catalyzed cleavage of the RNA analogue HpPNP. The pH-rate profile for this cleavage reaction showed that there is optimal catalytic activity at high pH, where the catalyst is in the basic form [Zn2(1)(HO-)]. However, it was also shown that the active form of the catalyst is Zn2(1)(H2O) and that this recognizes the C2-oxygen-ionized substrate in the cleavage reaction. The active catalyst Zn2(1)(H2O) shows a high affinity for oxyphosphorane transition state dianions and a stable methyl phosphate transition state analogue, compared with the affinity for phosphate monoanion substrates. The transition state binding energies, Delta G(S)(#), for cleavage of HpPNP catalyzed by a variety of Zn2+ and Eu3+ metal ion complexes reflect the increase in the catalytic activity with increasing total positive charge at the catalyst. These values of Delta G(S)(#) are affected by interactions between the metal ion and its ligands, but these effects are small in comparison with Delta G(S)(#) observed for catalysis by free metal ions, where the ligands are water. Enzymes are unique in having evolved mechanisms to effectively utilize binding interactions with nonreacting fragments of the substrate in stabilization of the reaction transition state. Orotidine 5'-monophosphate decarboxylase, alpha-glycerol phosphate dehydrogenase, and triosephosphate isomerase catalyze dissimilar decarboxylation, hydride transfer, and proton transfer reactions, respectively. Each enzyme derives ca. 12 kcal/mol of transition state stabilization from protein interactions with the nonreacting phosphate group, which is larger than the highest approximately 10 kcal/mol transition state stabilization that we have determined for small-molecule catalysis of phosphate diester cleavage in water. Each of these enzymes catalyze the slow reaction of a truncated substrate that lacks the phosphate group, and in each case, the reaction of the truncated substrate is strongly activated by the allosteric binding of the second substrate "piece" phosphite dianion, HPO3(2-). We propose a modular design for these enzymes with a classical active site that recognizes the reactive substrate fragment and a separate phosphodianion binding site. The second site is created, in part, by flexible protein loops that wrap around the substrate phosphodianion group and bury the substrate in an environment with an optimal local dielectric constant for the catalyzed reaction and with the most favorable positioning of the catalytic side chains. This design is easily generalized to a wide variety of enzyme-catalyzed reactions.

Zinc(II) Complexes as Hydrolytic Catalysts of Phosphate Diester Cleavage: From Model Substrates to Nucleic Acids

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Cleavage of phosphate diesters mediated by Zn(II) complex in Gemini surfactant micelles

The cleavage of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) catalyzed by the Zn(II)–biap (biap: N , N -bis(2-ethyl-5-methylimidazole-4-ylmethyl)aminopropane) complex has been investigated spectrophotometrically in a micellar solution of cationic Gemini surfactant 16-2-16 [bis(hexadecyldimethylammonium)ethane bromide] and CTAB (hexadecyltrimethylammonium bromide) at 25 ± 0.1 ° C . The experimental results reveal that a higher rate of acceleration (about 2016-fold) of HPNP cleavage promoted by the Zn(II)–biap complex has been observed in the 16-2-16 micellar solution in comparison with the background rate ( k 0 ) of HPNP spontaneous cleavage at 25 °C. Reaction rates of HPNP cleavage in CTAB micellar solutions are only about 40% of that in Gemini 16-2-16 micelles under comparable conditions. In addition, the cleavage rates of HPNP in Gemini micelles and in CTAB micelles are respectively 29.5 times and 12 times faster than that in aqueous buffer. Especially, a “sandwich absorptive mode” has been proposed to explain the acceleration of HPNP cleavage in a cationic micellar solution.

A transition state analog for phosphate diester cleavage catalyzed by a small enzyme-like metal ion complex

The values of K i for methylphosphate dianion ( MP 2−) inhibition of the cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate ( HpPNP) catalyzed by 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane ( Zn 2( 1)( H 2 O)) approach a small limiting value of 6 μM at pH < p K a = 7.8 for deprotonation of the catalyst to form Zn 2( 1)( HO −). There is a 1600-fold difference in the affinity of a phosphate diester monoanion (diethylphosphate) and phosphate monester dianion ( MP 2−) for Zn 2( 1)( H 2 O). This suggests that the latter is an analog for the transition state dianion for the cleavage reaction of HpPNP and other phosphate diesters. The observation that this transition state analog binds selectively to Zn 2( 1)( H 2 O) provides strong evidence that this is the active form of the catalyst which binds selectively to the ionized substrate. The efficiency of catalysis of the cleavage of phosphate diesters by Zn 2( 1)( H 2 O) and proteins is compared.

Mimicking Enzymes: Cooperation between Organic Functional Groups and Metal Ions in the Cleavage of Phosphate Diesters

A series of ligands derived from the bis-2-pyridinylmethylamine structure, which bear either additional hydroxyl or aromatic amino groups, were prepared and their Zn(II) complexes were studied as catalysts for the cleavage of bis-p-nitrophenyl phosphate (BNP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) diesters. A comparative kinetic study indicated that the insertion of organic groups, capable of acting as nucleophiles or as hydrogen-bond donors, substantially increases the hydrolytic activity of the metal complex. Dissection of the effects of the individual groups revealed that the increase in reactivity can reach up to three orders of magnitude. The improved efficiency of the systems studied, combined with the benefits resulting from the low pK(a) value of the active nucleophile, result in an acceleration of the BNP cleavage at pH 7 of six orders of magnitude. The pH-dependent reactivity profiles follow a bell-shaped curve and the maximum reactivity is observed at pH 9. The mechanism of the reactions and the structure of the complexes were investigated in detail by means of kinetic analysis, NMR spectroscopy experiments, and theoretical calculations. The reactivity of the complexes that cleave HPNP closely resembles the reactivity observed for BNP, but the accelerations achieved are lower as a result of different reaction mechanisms.

Zinc(ii) complexes as hydrolytic catalysts of phosphate diester cleavage: from model substrates to nucleic acids

The development of synthetic agents able to hydrolytically cleave phosphate diester bonds with high efficiency is a fascinating challenge, which will ultimately open the way to artificial nucleases able to compete with the natural enzymes. This Perspective highlights the progress reported in the realization of hydrolytic catalysts based on the Zn2+ ion, a metal ion which, due to its peculiar properties, is a very promising candidate. The review critically examines the reactivity of such catalysts toward model substrates and nucleic acids, paying particular attention to the strategies that can be pursued to improve efficiency and sequence selectivity.

Effect of Zn⋅⋅⋅Zn Separation on the Hydrolytic Activity of Model Dizinc Phosphodiesterases

From the study of highly preorganized model systems, experimental support has been obtained for a possible functional role of the Zn-(H)O...HO(H)-Zn motif in oligozinc hydrolases. The mechanistic relevance of such an array, which may be described as a hydrated form of a pseudo-terminal Zn-bound hydroxide, has recently been supported by DFT calculations on various metallohydrolase active sites. In the present targeted approach, the Zn...Zn distance in two related dizinc complexes has been controlled through the use of multifunctional pyrazolate-based ligand scaffolds, giving either a tightly bridged Zn-O(H)-Zn or a more loosely bridged Zn-(H)O...HO(H)-Zn species in the solid state. Zn-bound water has been found to exhibit comparable acidity irrespective of whether the resulting hydroxide is supported by strong hydrogen-bonding in the O(2)H(3) moiety or is in a bridging position between two zinc ions, indicating that water does not necessarily have to adopt a bridging position in order for its pK(a) to be sufficiently lowered so as to provide a Zn-bound hydroxide at physiological pH. Comparative reactivity studies on the cleavage of bis(4-nitrophenyl)phosphate (BNPP) mediated by the two dizinc complexes have revealed that the system with the larger Zn...Zn separation is hydrolytically more potent, both in the hydrolysis and the transesterification of BNPP. The extent of active site inhibition by the reaction products has also been found to be governed by the Zn...Zn distance, since phosphate diester coordination in a bridging mode within the clamp of two zinc ions is only favored for Zn...Zn distances well above 4 A. Different binding affinities are rationalized in terms of the structural characteristics of the product-inhibited complexes for the two different ligand scaffolds, with dimethyl phosphate found as a bridging ligand within the bimetallic pocket.

Structure-activity studies on the cleavage of an RNA analogue by a potent dinuclear metal ion catalyst: effect of changing the metal ion.

Dinuclear Cd(II), Cu(II), and Zn(II) complexes of L2OH (L2OH = 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane) are compared as catalysts for cleavage of the RNA analogue HpPNP (HpPNP = 2-hydroxypropyl 4-nitrophenyl phosphate) at 25 degrees C, I = 0.10 M (NaNO(3)). Zn(II) and Cu(II) readily form dinuclear complexes at millimolar concentrations and a 2:1 ratio of metal ion to L2OH at neutral pH. The dinuclear Zn(2)(L2O) and Cu(2)(L2O) complexes have a bridging alkoxide group that brings together the two cations in close proximity to facilitate cooperative catalysis. Under similar conditions, the dinuclear complex of Cd(II) is a minor species in solution; only at high pH values (pH 10.4) does the Cd(2)(L2O) complex become the predominant species in solution. Analysis of the second-order rate constants for cleavage of HpPNP by Zn(2)(L2O) is straightforward because a linear dependence of pseudo-first-order rate constant on dinuclear complex is observed over a wide pH range. In contrast, plots of pseudo-first-order rate constants for cleavage of HpPNP by solutions containing a 2:1 ratio of Cd(II) to L2OH as a function of increasing L2OH are curved, and second-order rate constants are obtained by fitting the kinetic data to an equation for the formation of the dinuclear Cd(II) complex as a function of pH and [L2OH]. Second-order rate constants for cleavage of HpPNP by these dinuclear complexes at pH 9.3 and 25 degrees C vary by 3 orders of magnitude in the order Cd(2)(L2O) (2.8 M(-)(1) s(-)(1)) > Zn(2)(L2O) (0.68 M(-)(1) s(-)(1)) > Cu(2)(L2O) (0.0041 M(-1) s(-1)). The relative reactivity of these complexes is discussed in terms of the different geometric preferences and Lewis acidity of the dinuclear Zn(II), Cu(II), and Cd(II) complexes, giving insight into the importance of these catalyst properties in the cleavage of phosphate diesters resembling RNA.