Article

Phosphodiester cleavage by trivalent lanthanides in the presence of native cyclodextrins

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Abstract

Testing phosphodiesterase activity of Eu(III) in the presence of native cyclodextrins revealed capacity of β-cyclodextrin (β-CD) to stabilize catalytically active metal hydroxocomplexes in mildly basic solutions. Kinetics of the hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) and transesterification of 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) as models of DNA and RNA respectively has been studied with La(III), Pr(III), Nd(III), Eu(III), Gd(III) and Dy(III) cations in the presence of β-CD in the range of pH 7.0-9.0. The overall catalytic effect with 2 mM lanthanide-β-CD complexes was up to 105 for HPNP and 108 for BNPP at pH 8 demonstrating the highest catalytic activity among so far reported artificial phosphodiesterases. Analysis of concentration and pH-dependences of observed rate constants for different lanthanides showed that active species are binuclear polyhydroxocomplexes of general type [M2(β-CD)(OH)n]6-n with n = 3-5. The metal-β-CD and phosphodiester-β-CD interactions were studied by 1H NMR spectroscopy. Mechanistic implications of much higher catalytic efficiency in BNPP hydrolysis as compared to HPNP transesterification are discussed.

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Article
In the present study, we apply a computational approach, based on DFT calculations and molecular dynamics simulations, to investigate the catalytic behavior of four supramolecular catalysts active in the cleavage of phosphodiesters. The QM data indicate the operation of a synchronous associative mechanism with limited differences in the structures and energies of the transition states. The comparison with the experimental data, expressed in terms of effective molarity (EM), suggests that the difference in catalytic performance are not ascribable to a difference in the enthalpy of activation. On the other hand, the analysis of the Molecular Dynamics trajectories clearly indicates the conformational mobility, and therefore the conformational entropy, to be at the origin of the superior catalytic efficiency of the catalysts based on a more preorganized structure. Essentially, the combined method presented here provides, at a limited computational cost, a tool that is simple, general, and potentially suited for a large variety of catalytic systems to rationalize their performances and to predict those of enzyme mimics to be synthesized in a rational design process. A method based on DFT calculations and Molecular Dynamics simulations aimed at determining the conformational entropy provides a tool to predict and rationalize experimental kinetic data in artificial phosphodiesterases. This approach can be generalized to a large number of bi‐ and multifunctional supramolecular catalysts and allow an a priori evaluation of the potential performance of a molecular scaffold.
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Chapter
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Dinuclear metallohydrolases are ubiquitously involved in biological transformations. For understanding and application purpose, significant efforts have been dedicated to the structural or/and functional mimics. Herein, a dinuclear Co(II) complex, Co2L, with intramolecular β-CDs is presented. The β-CDs confine a hydrophobic microenvironment that contributes to the valence stability of the ligated Co(II) ions. Species of Co2L in aqueous solution is clarified according to the results of potentiometric titration and UV–vis titration experiments. A highly positive-charged species [Co2HL]4+ is present at quasi-neutral condition, which contributed significantly to the binding and activity toward bis(4-nitrophenyl) phosphate (BNPP) assisted by the hydrophobic interactions exerted by intramolecular β-CDs. The kcat/KM value of Co2L is determined to be 9.8 × 10−2 M−1 s−1 at pH 6.5 and 308 K, which exceeds that of the reported zinc analog (Zn2L) by three orders of magnitude. Unexpectedly, Co2L also catalyzes hydrolysis of a phosphate monoester, 4-nitrophenyl phosphate (NPP). The observed phosphate esterase activities are rarely reported among synthetic Co(II) complexes.
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The rates and products of alkaline hydrolysis of a series of esters of 2-hydroxypropylphosphate have been studied. Decomposition by an epoxide route is dominant in the cyclohexyl and absent in the phenyl ester, in which hydrolysis occurs through the cyclic phosphate only. A linear relationship between log k and the pKa of the displaced alcohol is found to hold for the latter process, over a wide range of k and Ka values.
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The effects of α-, β-, and γ-cyclodextrin (CD) on intramolecular excimer formation between the two phenyl groups of diphenyl phosphate (DP) in aqueous solution were investigated by means of absorption and fluorescence spectra. It was found that DP forms 1 : 1 inclusion compounds with α-, β-, and γ-CDs. In the inclusion compound with α- or β-CD, only one phenyl group of a DP molecule is incorporated in the cavity of a CD molecule. In the case of γ-CD, however, the cavity can accommodate either one or two phenyl groups of a DP molecule. The pH dependence of the equilibrium constant for the formation of the inclusion compound between DP and β-CD has also been examined. Over the pH range from 3.46 to ≈10 the equilibrium constant does not change, whereas for pH values above ≈11 it decreases drastically.
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Unusual second-order, in metal, kinetics were observed for the hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) and 4-nitrophenyl diphenylphosphate (NPDPP) catalyzed by Y(III) in the presence of Bis-Tris propane (BTP) and Tris ligands at 25 °C in weakly basic aqueous solutions. Potentiometric and 1H-NMR titrations of BTP in the presence of Y(III) indicate formation of the mononuclear Y(BTP)3+ complex and dinuclear hydroxo complexes Y2(BTP)(OH)n6 − n where n = 2, 4, 5 or 6. Titrations of Tris in the presence of Y(III) were limited by the low stability of the system and were fitted to a tentative model involving formation of Y(Tris)3+ and a single dinuclear complex Y2(Tris)2(OH)5+. Comparison of concentration and pH-dependences of the reaction rates with the species distribution diagrams show that the catalytic hydrolysis of BNPP involves a simultaneous interaction of the substrate with Y2(BTP)(OH)42+ and Y2(BTP)(OH)5+ species in the Y(III)/BTP system and interaction with two Y2(Tris)2(OH)5+ species in the Y(III)/Tris system. The half-life for the hydrolysis of BNPP is only ca. 5 min at 25 °C and pH 8.5 in the presence of 4 mM Y(III) and 20 mM BTP. A rate enhancement by Y(III) in the hydrolysis of triester NPDDP is much smaller than that for diester BNPP. The hydrolysis of mono 4-nitrophenyl phosphate, the intermediate in BNPP hydrolysis, is zero-order in Y(III) indicating Michaelis–Menten type “saturation” kinetics.
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Cyclodextrins form complexes with lanthanide ions in basic aqueous solutions. This complex formation in basic solution dramatically enhances the solubility of lanthanide ions, which are otherwise insoluble due to the formation of hydroxide gels. Solutions of the ?-cyclodextrin-Ce3+ complex effectively hydrolyze 2'-deoxyadenosine-5'-monophosphate to 2'-deoxyadenosine.
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A few years ago, the remarkable catalytic activity of lanthanide ions for the hydrolysis of nucleic acids was discovered. With CeIV, DNA was hydrolysed under physiological conditions. For RNA hydrolysis, the last three lanthanide ions (TmIII, YbIII, and LuIII) are superb. Furthermore, artificial restriction enzymes for site-selective scission of DNA and RNA, essential tools for the future biotechnology, have been prepared by using the lanthanide complexes. The present article emphasizes the mechanistic aspects of the catalyses of these metal ions. Both DNA hydrolysis and RNA hydrolysis involve the cooperation of acid catalysis (by metal ion and/or metal-bound water) and base catalysis (by metal-bound hydroxide). The magnitudes of contributions of these catalyses, as well as the positions where they work, are primarily governed by the relative height of the energy-barrier for the formation of the pentacoordinated intermediate and that for its breakdown. The following conclusions have been obtained on the basis of various kinetic and spectroscopic evidence: (1) for the hydrolysis of both DNA and RNA, the catalytically active species are dinuclear hydroxo-clusters, (2) CeIV enormously activates DNA and promotes the formation of the pentacoordinated intermediate, and (3) the catalysis for RNA hydrolysis is mainly ascribed to the promotion of breakdown of the pentacoordinated intermediate.
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The effects of four Co(III) complexes, [Co(trien)(OH)(OH2)]2+, [Co(en)2(OH)(OH2)]2+, [Co(dien)(OH)(OH2)2]2+, and [Co(en)2(OH)(NH3)]2+, on the rate of hydrolysis of bis(p-nitrophenyl) phosphate, p-nitrophenyl phosphate, and bis(2,4-dinitrophenyl) phosphate have been examined at 50°C, under neutral-pH conditions. In neutral water, the cobalt-bound phosphodiester [Co(en)2(OH)[OP(O)(OC6H4NO2) 2]]+ is cleaved 107 times more rapidly (27 × 10-3 s-1) than the unbound phosphodiester, whereas the corresponding cobalt-bound phosphomonoester [Co(en)2(OH)[OP(O)2OC6H4NO 2]] is cleaved only 104 times more rapidly than the unbound phosphomonoester. The reactivity patterns for cobalt complex promoted hydrolysis of bis(p-nitrophenyl) phosphate and bis(2,4-dinitrophenyl) phosphate are, respectively, [Co(trien)(OH)(OH2)]2+ > [Co(en)2(OH)(OH2)]2+ > [Co(dien)(OH)(OH2)2]2+ and [Co(dien)(OH)(OH2)2]2+ > [Co(trien)(OH)(OH2)]2+ > [Co(en)2(OH)(OH2)]2+ > [Co(en)2(OH)(NH3)]2+. The difference in the reactivity pattern for the hydrolysis of the two phosphodiesters is explained in terms of a change in the rate-determining step with change in the phosphodiester reactivity. The structure of the amine ligand on the cobalt complex has a significant effect on both the rate of binding of the phosphate ester to the cobalt complex and the rate of cleavage of the phosphoester bond.
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The phosphate diestere, ethyl 4-nitrophenyl phosphate, and bis(4-nitrophenyl) phosphate in cis-[(en)2Ir(OH)O2P(OR)2]+ (en = 1,2-diaminoethane) react with the cis-hydroxo group at pH 8 to liberate nitrophenolate ion ∼106-fold faster than for the free ligand under the same conditions. The expected products of these reactions, the chelate phosphate esters, were not observed; only the ring-opened monodentate mpnoester products were obtained as a result of P-O bond cleavage. The cis-hydroxo ligand is a good nucleophile toward bound phosphate esters; however, the reactions of Ir(III) complexes described here proceed about 103-fold slower than the reactions of the analogous Co(III) complexes despite the more basic coordinated OH- on the iridium(III) ion. The reduction in rate is ascribed to the larger size of the Ir(III) ion compared to Co(III), which makes ring closure more difficult. While it is clear that coordinated OH- can be an effective nucleophile especially in an intramolecular reaction, it appears unlikely that reactions forming four-membered chelate phosphate esters will be relevant in biological systems because the biologically relevant ions Mg2+ and Zn2+ are relatively large ions (>Ir(III)). Also, the observation that the chelated ester prefers to undergo ring opening rather than lose the alcohol group implies that such chelate esters are unlikely to be effective in the enzymic systems.
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The efficiencies of three rigidly held cis-aquohydroxotetraazacobalt(III) complexes [(cyclen)Co(OH)(OH2)]2+, [(tren)Co(OH)(OH2)]2+, [(trpn)Co(OH)(OH2)]2+ in promoting the hydrolysis of bis(p-nitrophenyl)phosphate (BNPP) have been compared. In neutral water at 50°C, the rate constant for hydrolysis of the phosphate diester bond in [(cyclen)Co(OH)(BNPP)]+, [(tren)Co(OH)(BNPP)]+, [(trpn)Co(OH)(BNPP)]+ are 4.6 × 10-1, 8.1 × 10-3, and 2.5 s-1, respectively. [(trpn)Co(OH)(BNPP)]+ is hydrolyzed at about the same rate as BNPP bound to a real enzyme from Enterobacter aerogenes and about 1010 times more rapidly than free BNPP. The dramatic increase in the activity of the Co(III) complex with change in the tetraamine ligand structure can be explained in terms of a detailed mechanism of the reaction.
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The effect of alcohols on the beta-cyclodextrin (CD)/pyrene complex has been examined by using steady-state fluorescence measurements. A 1:1 stoichiometric ratio has been found between the alcohol and beta-CD. As the stoichiometry of the binary beta-CD/pyrene complex is 2:1, a ternary complex of stoichiometry of 2:1:2-beta-CD/pyrene/alcohol is proposed. Apparent formation constants in the presence of different alcohols have been determined by using the variation of the I/III vibronic band ratio of pyrene with increasing cyclodextrin concentration. The 2:1 beta-CD/pyrene stoichiometry for the binary complex has also been confirmed. The present study demonstrates that proper size matching among the pyrene, the cyclodextrin, and the alcohol leads to substantially larger equilibrium constants for the ternary complexes.
Article
Enormous rate accelerations are necessary to hydrolyze RNA (108-fold) or DNA (1017-fold) within minutes. There are three direct modes of activation that media ions can provide for hydrolyzing phosphate diesters. The rate accelerations due to Lewis acid activation (<102-fold), intramolecular nucleophile activation (108-fold), and leaving group activation (106-fold) may in some cases combine simply to give an overall rate acceleration in excess of 1016-fold; in other cases, greater cooperatively between the models of catalysis is possible. Double Lewis acid activation obtained by coordinating both phosphoryl oxygens of a phosphate diester to a variety of dinuclear metal complexes (Figures 2 and 3) can give far greater rate accelerations for the hydrolysis reaction (4 x 105-fold for a dinuclear Co(III) complex) than single Lewis acid activation (<102). This mode of activation is possible with an intermetal distance from 2.9 to 7.0 Å and is particularly useful for cleaving RNA efficiently. For DNA hydrolysis, double Lewis acid activation by itself is not enough for efficient cleavage as it is about 109 times more stable than RNA. The nucleophile activation (metal hydroxide, metal alkoxide, metal-bridging oxide, metal-bridging peroxide) is important for DNA hydrolysis but not for RNA cleavage as its 2'- OH group already acts as a highly efficient internal nucleophile (Figure 1). However, the metal-activated nucleophiles may in some cases act as general base catalysts in cleaving RNA. While all of the above-mentioned nucleophiles can cleave phosphates with good leaving groups, only metal hydroxides appear to cleave those with poor leaving groups. For the other nucleophiles to be effective (metal alkoxide, metal-bridging oxide, metal-bridging peroxide), leaving group activation (coordination of the poor leaving group oxygen to the metal) is required.
Article
Kinetics of transesterification of the RNA model substrate 2-hydroxypropyl 4-nitrophenyl phosphate promoted by Mg(2+) and Ca(2+), the most common biological metals acting as cofactors for nuclease enzymes and ribozymes, as well as by Co(NH(3))(6)(3+), Co(en)(3)(3+), Li(+), and Na(+) cations, often employed as mechanistic probes, was studied in 80% v/v (50 mol %) aqueous DMSO, a medium that allows one to discriminate easily specific base (OH(-)-catalyzed) and general base (buffer-catalyzed) reaction paths. All cations assist the specific base reaction, but only Mg(2+) and Na(+) assist the general base reaction. For Mg(2+)-assisted reactions, the solvent deuterium isotope effects are 1.23 and 0.25 for general base and specific base mechanisms, respectively. Rate constants for Mg(2+)-assisted general base reactions measured with different bases fit the Brønsted correlation with a slope of 0.38, significantly lower than the slope for the unassisted general base reaction (0.77). Transition state binding constants for catalysts in the specific base reaction (K(⧧)(OH)) both in aqueous DMSO and pure water correlate with their binding constants to 4-nitrophenyl phosphate dianion (K(NPP)) used as a minimalist transition state model. It was found that K(⧧)(OH) ≈ K(NPP) for "protic" catalysts (Co(NH(3))(6)(3+), Co(en)(3)(3+), guanidinium), but K(⧧)(OH) ≫ K(NPP) for Mg(2+) and Ca(2+) acting as Lewis acids. It appears from results of this study that Mg(2+) is unique in its ability to assist efficiently the general base-catalyzed transesterification often occurring in active sites of nuclease enzymes and ribozymes.
Article
Direct excitation europium(III) luminescence spectroscopy is used to study the speciation of aqueous europium(III) ions at micromolar concentrations and near neutral pH. The pH and concentration dependence of the europium(III) 7F0→5D0 excitation peak is consistent with the formation of both mononuclear and dinuclear europium(III) hydroxide complexes at pH 6.5. Luminescence intensity and lifetime quenching studies in the presence of NdIII at pH 5.0 and 6.5 support the formation of a dinuclear complex at pH 6.5. Steady state excitation and time-resolved luminescence spectroscopy are consistent with the formation of innersphere nitrate and fluoride complexes, but outersphere perchlorate and chloride complexes at pH 6.5 and 5.0.
Article
Conditional stability constants of 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (BT) complexes of trivalent rare earth element (Ln) ions (La, Nd, Eu, Gd, Yb, Dy, Er, Lu) and Y were determined potentiometrically in aqueous NaCl solutions at 30C and 0.1 M ionic strength. Least-squares fitting shows that, at 3+ is dominant, with LnBT2 3+ forming a secondary complex, where: Ln3 + + BT « LnBT3 + b11 Ln3 + + 2BT « Ln(BT)23 + b21 \begin{gathered} { Ln}^{{3 + }} + {BT} \leftrightarrow {LnBT}^{{3 + }} { }\beta _{11} \hfill \\ {Ln}^{{3 + }} + 2{BT} \leftrightarrow {Ln(BT)}_{2}^{{3 + }} { }\beta _{21} \hfill \\ \end{gathered} Conditional stability constants appear to be directly related to the ionic radius of the trivalent ion in question. The optimal ionic radius, 104–105 pm, yields values of log b21* = 10.93 ±0.63\beta _{{21}}^{*} = 10.93 \pm 0.63 (Gd) and b11* = 6.83 ±0.14\beta _{{11}}^{*} = 6.83 \pm 0.14 (Yb). Complexation drops off steeply on either side of the ideal ionic radius. In relating the stability constants to ionic radius, it is assumed that BT complexes with Gd, Dy, Er, and Lu have coordination number eight, whereas those with La, Nd, and Eu have coordination number nine. The smoothest trend of stability constants with ionic radius is obtained if Yb–BT complexes are assumed to have coordination number nine. These results may reflect the ability of BT to form an ionic radius-specific chelate structure.
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The review presents a survey of the metal complexing properties of native cyclodextrins (including deprotonation in alkaline medium) and a report on some recent results on composition and stability of metal–cyclodextrin complexes.
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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.
Article
Two N-donor ligands (L(1) and L(2)) derived from a β-cyclodextrin (βCD) monomer and dimer were employed to mediate the hydrolytic activity and stability of the Ce(IV) ion in aqueous solution. Complexes Ce(IV)-L(1) and Ce(IV)-L(2) were prepared in situ and characterized by means of UV-vis and NMR measurements. Ce(IV)-L(1) catalyzed the hydrolysis of a DNA model, bis(4-nitrophenyl)phosphate (BNPP) with k(cat) = 5.2 × 10(-3) s(-1) (half-life t(1/2) ≈ 2 minutes) under mild conditions, which represented an approximate 130 million-fold acceleration with respect to the spontaneous hydrolysis of BNPP. The dinuclear species, [Ce(2)L(1)(2)(OH)(5)](3+), contributed splendidly to the catalytic efficiency which echoed the active species postulation of [Ce(2)(OH)(7)](+) in the literature. Ce(IV)-L(2) exhibited efficient binding with BNPP giving 1/K(M) = 2.1 × 10(5) M(-1) which exceeded other Ce(IV) species, e.g. [Ce(4)(OH)(15)](+), by 2 orders of magnitude, which highlighted the hydrophobicity effect of βCDs. Such a highly binding affinity leads to the second-order rate constant, k(cat)/K(M) = 2.3 × 10(2) M(-1) s(-1), which probably ranks as the highest in the non-enzymatic cleavage of BNPP under similar conditions. Additionally, Ce(IV)-L(2) showed favorable tolerance to basic aqua owing to the bulky protection of double βCD pendants.
Article
The interaction of trivalent lanthanide ions (Ln3+) with native cyclodextrins (CDs) is investigated in acidic and basic aqueous media. At low pH, the association constants for 1:1 complexes are in the range logK=2–4 (μ=0.1 M, NaCl or TMACl), as determined by pH-potentiometric study of the hydrolysis of the Ln ions in absence and in presence of CDs. The thermodynamic parameters of the inclusion reaction show that the complexation of Ln3+ ions inside the CD cavity is entropically driven and does not depend upon the host. 1H and 13C NMR spectra point to inclusion of La3+ cations in the cavity of α-CD occurring in the narrowest part of the host molecule, close to the C5 carbon atoms. α-CD acts like a crown ether, its anomeric oxygen atoms being the donor atoms. Complexation of Tb3+ by partially deprotonated α-CD was investigated at pH 12.33: 13C NMR measurements show that complexation occurs at the OH-3 secondary hydroxyl groups of α-CD while UV–Vis spectrophotometric titration leads to an apparent constant logKapp≈4.2 (1:1 complex). Lifetime measurements of the Eu(5D0) and Tb(5D4) levels confirm these findings.
Article
Dinuclear europium(III) complexes of the macrocycles 1,3-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-m-xylene (1), 1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene (2), and mononuclear europium(III) complexes of macrocycles 1-methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (3), 1-[3'-(N,N-diethylaminomethyl)benzyl]-4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (4), and 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (5) were prepared. Studies using direct excitation ((7)F0 --> (5)D0) europium(III) luminescence spectroscopy show that each Eu(III) center in the mononuclear and dinuclear complexes has two water ligands at pH 7.0, I = 0.10 M (NaNO3) and that there are no water ligand ionizations over the pH range of 7-9. All complexes promote cleavage of the RNA analogue 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at 25 degrees C (I = 0.10 M (NaNO3), 20 mM buffer). Second-order rate constants for the cleavage of HpPNP by the catalysts increase linearly with pH in the pH range of 7-9. The second-order rate constant for HpPNP cleavage by the dinuclear Eu(III) complex (Eu2(1)) at pH 7 is 200 and 23-fold higher than that of Eu(5) and Eu(3), respectively, but only 7-fold higher than the mononuclear complex with an aryl pendent group, Eu(4). This shows that the macrocycle substituent modulates the efficiency of the Eu(III) catalysts. Eu2(1) promotes cleavage of a dinucleoside, uridylyl-3',5'-uridine (UpU) with a second-order rate constant at pH 7.6 (0.021 M(-1) s(-1)) that is 46-fold higher than that of the mononuclear Eu(5) complex. Methyl phosphate binding to the Eu(III) complexes is energetically most favorable for the best catalysts, and this supports an important role for the catalyst in stabilization of the developing negative charge on the phosphorane transition state. Despite the formation of a bridging phosphate ester between the two Eu(III) centers in Eu2(1) as shown by luminescence spectroscopy, the two metal ion centers are only weakly cooperative in cleavage of RNA and RNA analogues.
Article
Lanthanide(III) ions have shown enormous catalyses for the hydrolysis of the phosphodiester linkages in RNA, indicating their high potential for versatile applications to biotechnology and molecular biology. The activity monotonically increases with increasing atomic number in the lanthanide series, the last three ions (Tm3+, Yb3+, and Lu3+) being the most active. Non-lanthanide metal ions are virtually inactive. The pseudo first-order rate constant for the hydrolysis of adenylyl(3'-5')adenosine (ApA) by LuCl3 (5 mmol x dm(-3)) at pH 7.2 and 30 degrees C is 1.9 x 10(-1) min(-1) (the half-life is only 3.6 min), corresponding to 10(8)-fold acceleration. The product is an equimolar mixture of adenosine and its 2'- or 3'-monophosphate without any byproducts. The 2',3'-cyclic monophosphate of adenosine is not accumulated much in the reaction mixture. Lanthanide ions also efficiently hydrolyze oligoribonucleotides without a specific base-preference. In ApA hydrolysis by NdCl3 and GdCl3, the dependence of the hydrolysis rate on either the pH or concentration of the metal salt coincides fairly well with the corresponding profile of the equilibrium concentration of the bimetallic hydroxo-cluster [M2(OH)2]4+ (M=metal ion). Both the formation of the pentacoordinated intermediate and its decomposition are greatly promoted by lanthanide ions. A catalytic mechanism in which two metal ions (or their coordination water) in these tetracationic hydroxo-clusters show acid/base cooperation is proposed.
Article
The Zr(IV)-tetraphenylpor-phyrinates Zr(TPP)(X,X'), (X,X' = -OAc, -OMe, Cl ) 4-6, 8 were prepared and their complexing properties as well as catalytic properties towards solvolysis of the phosphate diesters hpp (2), dmp (3) and pmp (16) characterised. The diesters 2 and 16, representing model phosphates for RNA and DNA, were substrates for the catalyst Zr(TPP)Cl2 (4), and rate accelerations over background by 6-9 orders of magnitude were measured. These accelerations are comparable to those of dinuclear transition metal catalysts and lanthanide ions. Catalytic turnover was observed. Kinetic studies revealed that the catalytically active species of 4 in the solvolysis of 2 and 16 in methanol-containing solvents are dinuclear complexes containing either one or two phosphate esters depending upon the phosphate concentration. Besides the usual solvolysis pathway of the RNA model hpp (2), which proceeds via the cyclophosphate 20, a second, unusual pathway via direct substitution of the hydroxypropyl substituent was found. X-ray analysis of the Zr(TPP)(dmp) complex 19 revealed a dinuclear structure with two bridging dmp ligands and one monomethyl phosphate unit. In 19 one of the two dmp residues occurs in a very unusual high energy ac,ap conformation. Based on this structure and on the kinetic data, mechanistic models for the two solvolysis reaction pathways were developed. From an extensive CSD search on phosphodiester structures no correlation between P-O ester bond lengths and diester conformations could be found. However, P-O ester bonds decrease in length with increasing formal charge of the complexing metal ions. This underlines the higher importance of electrostatic activation relative to stereoelectronic effects in phosphodiester hydrolysis.
Article
Lanthanide ions are remarkably effective catalysts for the hydrolytic cleavage of phosphate ester bonds, including the robust bonds of DNA. This makes Ln(III) and Ce(IV) ions attractive candidates for developing selective and efficient artificial nucleases, which could have many biochemical and clinical applications. Both small-molecule-based and biopolymer-based lanthanide complexes are being pursued.
Article
Potentiometric titrations of the mixtures of lanthanide(III) perchlorates and bis-Tris propane (BTP) reveal formation of dinuclear hydroxo complexes M2(BTP)2(OH)n(6-n), where M = La(III), Pr(III), Nd(III), Eu(III), Gd(III), and Dy(III) and n = 2, 4, 5, or 6, in the pH range 7-9. ESI-MS data confirm the presence of dinuclear species. Kinetics of the hydrolysis of bis(4-nitrophenyl) phosphate (BNPP), mono-4-nitrophenyl phosphate (NPP), and 4-nitrophenyl acetate (NPA) in the lanthanide(III)-BTP systems has been studied at 25 degrees C in the pH range 7-9. The second-order rate constants for the hydrolysis of BNPP by individual lanthanide hydroxo complexes have been estimated by using the multiple regression on observed rate constants obtained at variable pH. For a given metal, the rate constants increase with increasing in the number n of coordinated hydroxide ions. In a series of complexes with a given n, the second-order rate constants decrease in the order La > Pr > Nd > Eu > Gd > Dy. Hydrolysis of NPP follows Michaelis-Menten-type "saturation" kinetics. This difference in kinetic behavior can be attributed to stronger binding of NPP dianion than BNPP monoanion to the lanthanide(III) species. Activities of lanthanide complexes in the hydrolysis of NPA, which is 10(6) times more reactive than BNPP in alkaline or aqueous hydrolysis, are similar to those in BNPP hydrolysis indicating unique capability of lanthanide(III) cations to stabilize the transition state of phosphate diester hydrolysis. Results of this study are analyzed together with literature data for other metal cations in terms of the Brønsted correlation and transition state-catalyst complexation strength.
Article
The methanolysis of hydroxypropyl-p-nitrophenyl phosphate (HPNPP, 1) promoted by La(OTf)(3) under buffered conditions was studied in methanol as a function of pH at 25 degrees C. (31)P NMR studies at -90 degrees C indicate that there are at least three La/1 complexes formed at pH approximately 5.3 of 1:1, 2:2, and 1:2 stoichiometry. Kinetic studies of the observed pseudo-first-order rate constants for the methanolysis of 1 as a function of [La(3+)] at 4.5 < pH < 10.5 indicate there are two general pH regimes. In the low pH regime between 4.5 and 7.6, the plots of k(obs) versus [La(3+)] exhibit saturation behavior with very strong 1:1 binding, with a plateau rate constant that depends on [OCH(3)(-)]. The catalytically productive species is shown to be a 2:2 complex of La(3+) and 1, where the phosphate is proposed to be doubly activated, thereby promoting the methoxide reaction by some 4.6 x 10(10)-fold. In the high pH regime from 7.9 to 10.5, 1:1, 2:2, and 2:1 La(3+)/1 complexes are formed with the La(3+) coordinated in the form of [La(3+)(OCH(3)(-))](1,2). Throughout this pH regime at high [La(3+)], a saturation complex, (La(3+)OCH(3)(-))(2)/1, is formed that spontaneously decomposes with a rate constant of (5-10) x 10(-)(3) s(-)(1), leading to an acceleration of 10(9)-fold at pH 8.0.
Article
The catalytic effects of the Zn(II) complexes of a series of poliaminic ligands in the hydrolysis of the activated phosphodiesters bis-p-nitrophenyl phosphate (BNP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) have been investigated. The reactions show first-order rate dependency on both substrate and metal ion complex and a pH dependence which is diagnostic of the acid dissociation of the reactive species. The mechanism of the metal catalyzed transesterification of HPNP has been assessed by solvent isotopic kinetic effect studies and involves the intramolecular nucleophilic attack of the substrate alcoholic group, activated by metal ion coordination. The intrinsic reactivity of the different complexes is controlled by the nature and structure of the ligand: complexes of tridentate ligands, particularly if characterized by a facial coordination mode, are more reactive than those of tetradentate ligands which can hardly allow binding sites for the substrate. In the case of tridentate ligands that form complexes with a facial coordination mode, a linear Brønsted correlation between the reaction rate (log k) and the pK(a) of the active nucleophile is obtained. The beta(nuc) values are 0.75 for the HPNP transesterification and 0.20 for the BNP hydrolysis. These values are indicated as the result of the combination of two opposite Lewis acid effects of the Zn(II) ion: the activation of the substrate and the efficiency of the metal coordinated nucleophile. The latter factor apparently prevails in determining the intrinsic reactivity of the Zn(II) complexes.
Article
The cyclization/cleavage of 3',5'-uridyluridine to form 2',3'-cyclic uridylic acid is very effectively catalyzed by Eu3+, and the cyclization/cleavage of the 1-p-nitrophenyl phosphate ester of propane-1,2-diol also shows strong metal ion catalysis by Eu3+, Tb3+, and Yb3+. It also shows moderate catalysis by Mg2+, but not by Ca2+; Zn2+ and Pb2+ are also good catalysts. Various ligands activate these reactions further, and imidazole apparently acts as an additional base catalyst. Some cyclodextrin derivatives act to bind both the substrate and the metal ion but, contrary to what is reported elsewhere, there is no strong selectivity among nucleotides that can be ascribed to cyclodextrin binding.
Article
Glycine and N,N-dimethylglycine stabilize La(III) hydroxide complexes of the type La2L2(OH)4 which possess phosphodiesterolytic activity close to that observed with most active tetravalent cations like Ce(IV).
Article
The trivalent lanthanide ions are chemically similar to Ca(II) ions, making them useful Ca analogs for a multitude of applications. In addition, Ln(III) ions are efficient catalysts of hydrolysis due to their much stronger Lewis acidity relative to Ca(II) ions. Ln-binding peptides thus offer both the opportunity to study known Ca sites as well as to explore new biological functions with an entire family of spectroscopically rich and reactive ions. This review discusses Ln-binding peptides in three roles: (i) as models of Ca-protein structure and function, (ii) as spectroscopic tags for protein expression and characterization and (iii) as designed artificial endonucleases. The creation of hydrolytically active Ln peptides that can fold, bind, cleave and discriminate among substrates shows that the design of Ln enzymes can be accomplished, and they will serve as versatile biochemical tools to investigate protein folding, structure and nuclease function.
Article
Using per(3,6-anhydro)cyclodextrin derivatives [per(3,6-anhydro)CD], it was possible to produce new lanthanide chelates by careful choice of the size and functional groups. Heptakis(3,6-anhydro-2-O-methyl)cyclomaltoheptaose fulfils the best criteria for complexation of lanthanide ions. Nuclear magnetic resonance was used to derive the association constants and the stoichiometries of these new complexes. Finally, a three-dimensional structure of these complexes consistent with the NMR data is proposed, to ascertain the position of lanthanide in the cavity of the per(3,6-anhydro)CD. For the present purposes, heptakis(2-O-acetyl-3,6-anhydro)cyclomaltoheptaose, octakis(2-O-acetyl-3,6-anhydro)cyclomaltooctaose, heptakis(3,6-anhydro-2-O-methyl)cyclomaltoheptaose and octakis(3,6-anhydro-2-O-methyl)cyclomaltooctaose have been synthesized and purified.
Article
The effect of increasing pL on the extraordinary catalytic activity of a dinuclear Zn2+ complex toward cleavage of uridine 3'-4-nitrophenyl phosphate (UpPNP) in H2O and D2O was determined. This change from H2O to D2O causes an increase from 7.8 to 8.4 in the apparent pKa of a catalytic functional group, but has little effect on the activity of the active form of the catalyst toward cleavage of UpPNP, so that there is no primary kinetic SDIE on the cleavage reaction from movement of a proton at the rate-determining transition state. It is concluded that essentially all of the rate acceleration for this catalyst is due to electrostatic stabilization of the transition state by interactions between opposing cationic and anionic charges.
Article
This review deals with enzymes where structures and kinetic studies have provided information on chemical mechanism and transition-state structure. Emphasis is put on nonenzymatic phosphate transfer, enzymatic phosphate transfer and sulfate transfer. Under nonenzymatic phosphate transfer, discussion covers uncatalyzed reactions of phosphomonoesters, reactions of phosphodiesters and implications for enzymatic catalysis. As for enzymatic phosphate transfer, emphasis is on phosphatases, regulatory enzymes, phosphoglucomutases, kinases, enzymes with a carboxyphosphate intermediate, transcarbomylases, ATPases and ATP synthesis, phosphodiesterases, nucleotidyltransferases and phosphotriesterase. With regards to sulfate transfer, topics discussed include types of sulfate esters, uncatalyzed reactions of sulfomonoesters, uncatalyzed reactions of sulfate diesters and enzymatic sulfuryl transfer from PAPS.
Article
(Figure Presented) A combined attack: Hydrogen-bonding interactions with double Lewis acid activation generate a dinuclear ZnII complex that is exceptionally effective for binding monoanionic phosphate diesters in water and for catalyzing phosphodiester transesterifications. The complex catalyzes the hydrolytic cleavage of RNA-like activated, artificial substrates and nonactivated, natural substrates with similar efficiencies.
Article
The anion of 4-imidazolecarboxylic acid (HL) stabilizes hydroxo complexes of trivalent lanthanides of the type ML(OH)+ (M = La, Pr) and M2L(n)(OH)(6-n) (M = La, n = 2; M = Pr, n = 2, 3; M = Nd, Eu, Dy, n = 1-3). Compositions and stability constants of the complexes have been determined by potentiometric titrations. Spectrophotometric and (1)H NMR titrations with Nd(III) support the reaction model for the formation of hydroxo complexes proposed on the basis of potentiometric results. Kinetics of the hydrolysis of two phosphate diesters, bis(4-nitrophenyl) phosphate (BNPP) and 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP), and a triester, 4-nitrophenyl diphenyl phosphate (NPDPP), in the presence of hydroxo complexes of five lanthanides were studied as a function of pH and metal and ligand concentrations. With all lanthanides and all substrates, complexes with the smallest n, that is M2L2(OH)4 for La and Pr and M2L(OH)5 for Nd, Eu, and Dy, exhibited the highest catalytic activity. Strong inhibitory effects by simple anions (Cl-, NO3-, (EtO)2PO2-, AcO-) were observed indicating high affinity of neutral hydroxo complexes toward anionic species. The catalytic activity decreased in the order La > Pr > Nd > Eu > Dy for both diester substrates and was practically independent of the nature of cation for a triester substrate. The efficiency of catalysis, expressed as the ratio of the second-order rate constant for the ester cleavage by the hydroxo complex to the second-order rate constant for the alkaline hydrolysis of the respective substrate, varied from ca. 1 for NPDPP to 10(2) for HPNPP and to 10(5) for BNPP. The proposed mechanism of catalytic hydrolysis involves reversible bridging complexation of a phosphodiester to the binuclear active species followed by attack on the phosphoryl group by bridging hydroxide (BNPP) or by the alkoxide group of the deprotonated substrate (HPNPP).
Article
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.
Article
The catalytic ability of a dinuclear Zn2+ complex of 1,3-bis-N1-(1,5,9-triazacyclododecyl)propane (3) in promoting the cleavage of an RNA model, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP, 1), and a DNA model, methyl p-nitrophenyl phosphate (MNPP, 4), was studied in methanol solution in the presence of added CH3O- at 25 degrees C. The di-Zn2+ complex (Zn2 :3), in the presence of 1 equiv of added methoxide, exhibits a second-order rate constant of (2.75 +/- 0.10) x 10(5) M(-1) s(-1) for the reaction with 1 at s(s)pH 9.5, this being 10(8)-fold larger than the k2 value for the CH3O- promoted reaction (kOCH3 = (2.56 +/- 0.16) x 10(-3) M(-1) s(-1)). The complex is also active toward the DNA model 4, exhibiting Michaelis-Menten kinetics with a KM and kmax of 0.37 +/- 0.07 mM and (4.1 +/- 0.3) x 10(-2) s(-1), respectively. Relative to the background reactions at s(s)pH 9.5, Zn2 :3 accelerates cleavage of each phosphate diester by a remarkable factor of 1012-fold. A kinetic scheme common to both substrates is discussed. The study shows that a simple model system comprising a dinuclear Zn2+ complex and a medium effect of the alcohol solvent achieves a catalytic reactivity that approaches enzymatic rates and is well beyond anything seen to date in water for the cleavage of these phosphate diesters.
Article
Potentiometric titrations of La(III), Nd(III), and Eu(III) perchlorates by Me 4N(OH) in 80% vol aq DMSO revealed formation of predominantly mononuclear complexes M(OH)n(3- n) (n = 1, 2, or 3) and a single binuclear complex M2(OH)(5+). Kinetics of the cleavage of two phosphate diesters, bis (4-nitrophenyl) phosphate (BNPP) and 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP), and a triester, 4-nitrophenyl diethyl phosphate (paraoxon), were studied as a function of metal and Me4N(OH) concentrations in the same medium. Rate of BNPP cleavage is second-order in metal and is proportional to the product of concentrations of M(OH)2(+) and M(OH)3 species. Rate of HPNPP cleavage is proportional to [M(OH)3](3) for La(III) and Nd(III) and to [M(OH)3](2) for Eu(III). Proposed mechanism for BNPP hydrolysis involves formation of M2(OH)5(diester) intermediate followed by intramolecular nucleophilic attack of hydroxide anion on the phosphoryl group of the substrate. Proposed mechanism for HPNPP cleavage involves formation of M3(OH)9(diester)(-) or M2(OH)6(diester)(-) intermediates followed by the general base-assisted intramolecular cyclization of HPNPP. The latter mechanism is supported by observation of the solvent kinetic isotope effect k H/kD = 2.9 for Eu(III) catalyzed HPNPP cleavage. The efficiency of catalysis in 80% DMSO is much higher than in water. The reaction rate observed in the presence of 1 mM metal in neutral solution surpasses the rate of background hydrolysis by a factor of 10(12)-10(13) for BNPP and 10(10) for HPNPP. The increased catalytic activity is attributed principally to the preferable solvation of lanthanide ions by DMSO, which creates an anhydrous microenvironment favorable for reaction in the coordination sphere of the catalyst. The catalytic activity of lanthanides in paraoxon hydrolysis is much lower with the estimated efficiency of catalysis about 10(5) for 1 mM La(III).
Article
Reactivity data related to processes in which molecular receptors promote the reaction of two simultaneously complexed reactants have been surveyed and analyzed in terms of effective molarity (EM). Methods and criteria for the calculation of reliable EM's have been highlighted. Extension of a previous extrathermodynamic treatment of intramolecular reactions of bifunctional chains to the intracomplex reactions of the ternary complexes involved in two-substrate catalyzed reactions has provided a sound framework for a comparative analysis of reactivity and catalytic efficiency in structurally diverse and apparently unrelated systems.
Article
A novel beta-cyclodextrin dimer, 1,10-phenanthroline-2,9-dimethyl-bridged-bis(6-monoammonio-beta-cyclodextrin) (phenBisCD, L), was synthesized. Its zinc complex (ZnL) has been prepared, characterized, and applied as a new catalyst for diester hydrolysis. The formation constant (logK(ML)=9.56+/-0.01) of the complex and deprotonation constant (pK(a)=8.18+/-0.04) of the coordinated water molecule were determined by a potentiometric pH titration at (298+/-0.1) K. Hydrolytic kinetics of carboxylic acid esters were performed with bis(4-nitrophenyl) carbonate (BNPC) and 4-nitrophenyl acetate (NA) as substrates. The obtained hydrolysis rate constants showed that ZnL has a very high rate of catalysis for BNPC hydrolysis, giving a 3.89x10(4)-fold rate enhancement over uncatalyzed hydrolysis at pH 7.01, relative to only a 42-fold rate enhancement for NA hydrolysis. Moreover, the hydrolysis second-order rate constants of both BNPC and NA greatly increases with pH. Hydrolytic kinetics of a phosphate diester catalyzed by ZnL was also investigated by using bis(4-nitrophenyl) phosphate (BNPP) as the substrate. The pH dependence of the BNPP cleavage in aqueous buffer shows a sigmoidal curve with an inflection point around pH 8.11, which was nearly identical to the pK(a) value from the potentiometric titration. The k(cat) of BNPP hydrolysis promoted by ZnL was found to be 9.9x10(-4) M(-1) s(-1), which is comparatively higher than most other reported Zn(II)-based systems. The possible intermediate for the hydrolysis of BNPP, BNPC, and NA catalyzed by ZnL is proposed on the basis of kinetic and thermodynamic analysis.