Bis 4-nitrophenyl Phosphate Research Articles

Dicopper(II) complexes showing DNA hydrolase activity and monomeric adduct formation with bis(4-nitrophenyl)phosphate

Ferromagnetic dicopper(II) complexes [Cu2(μ-O2CCH3)(μ-OH)(L)2(μ-L1)](PF6)2, where L=1,10-phenanthroline (phen), L1=H2O in 1 and L=dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), L1=CH3CN in 2, are prepared and structurally characterized. Crystals of 1 and 2 belong to the monoclinic space group of P21/n and P21/m, respectively. The copper(II) centers display distorted square-pyramidal geometry having a phenanthroline base and two oxygen atoms of the bridging hydroxo and acetate group in the basal plane. The fifth coordination site has weak axially bound bridging solvent molecule H2O in 1 and CH3CN in 2. The Cu···Cu distances are 3.034 and 3.046Å in 1 and 2, respectively. The complexes show efficient hydrolytic cleavage of supercoiled pUC19 DNA as evidenced from the mechanistic studies that include T4 DNA ligase experiments. The binuclear complexes form monomeric copper(II) adducts [Cu(L)2(BNPP)](PF6) (L=phen, 3; dpq, 4) with bis(4-nitrophenyl)phosphate (BNPP) as a model phosphodiester. The crystal structures of 3 and 4 reveal distorted trigonal bipyramidal geometry in which BNPP binds through the oxygen atom of the phosphate. The kinetic data of the DNA cleavage reactions of the binuclear complexes under pseudo- and true-Michaelis–Menten conditions indicate remarkable enhancement in the DNA hydrolysis rate in comparison to the control data.

Abstract 670: Bis-nitrophenylphosphate (BNPP) but not phenylmethylsulfonyl fluoride (PMSF) inhibits the activation of the novel anti-cancer pro-drug CZ48

Abstract CZ48, the 20-O-propionate ester of Camptothecin (CPT), is a non-toxic prodrug of CPT first described by Cao et al. (1998). The propionate side-chain is enzymatically cleaved in target tissues. This gives rise to CPT, an inhibitor of Topoisomerase I, which then blocks cell division causing selective tumor toxicity. We have previously established the preferential activation of CZ48 in tumor tissues compared to normal tissues and an ultra sensitive assay to detect it (2009). The present studies explore the specifics of that activation in vitro using both cells in culture and surgically removed xenografted tumors from nude mice. DOY lung adenocarcinoma was grown subcutaneously in nude mice and then surgically excised, homogenized and serially centrifuged to produce specific subcellular fractions. The bulk of esterase activity was found to be concentrated in the cytosol, while the microsomal fraction showed minimal activity. All other fractions tested were below the limit of detection. HT-29 colon carcinoma cells were grown in culture in the presence and absence of varying concentrations of bis-nitrophenylphosphate (BNPP), a potent inhibitor of carboxylesterase and butyrylcholinesterase (BChE), in an attempt to find the highest possible non-toxic concentration to the cells. This concentration was then included during a 7-day IC50 determination for CZ48. In the presence of BNPP, the IC50 of CZ48 increased 4-fold from 181.4 nM to 710.8 nM. In vitro conversion of CZ48 to CPT by cytosolic fraction of a HT-29 tumor xenograft grown subcutaneously in nude mice was decreased by BNPP in a dose dependent manner. In contrast, the serine protease and carboxylesterase inhibitor phenylmethylsulfonyl fluoride (PMSF) and other esterase inhibitors did not affect the activation of CZ48. Purified butyrylcholinesterase and acetylcholinesterase (SigmaAldrich, St. Louis MO) did not show any capacity to activate CZ48. In conclusion, the enzymatic activation of CZ48 is not mediated by butyrylcholinesterase but by another cytosolic enzyme inhibitable by BNPP. These data constitute a significant step forward in unraveling the exact mechanism for the activation of CZ48. This will help design a pre-treatment assay to establish tumor sensitivity, as well as give us direction for designing new, more potent prodrugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 670. doi:10.1158/1538-7445.AM2011-670

Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.

A novel asymmetric di-Ni(ii) system as a highly efficient functional model for phosphodiesterase: synthesis, structures, physicochemical properties and catalytic kinetics

A novel asymmetric phenol-based 'end-off' dinucleating ligand 2-{[(2-piperidylmethyl)amino]methyl}-4-bromo-6-[(1-methylhomopiperazine-4-yl)methyl]phenol (HL) and three dinuclear nickel(II) complexes, [Ni₂L(μ-OH)] (ClO₄)₂ (1), [Ni₂L(DNBA)₂(CH₃CN)₂]BPh₄ (2) and [Ni₂L(BPP)₂(CH₃CN)₂]BPh₄ (3) have been synthesized and characterized by a variety of techniques including: NMR, infrared and UV-vis spectroscopies, mass spectrometry, elemental analysis, molar conductivity, thermal analysis, magnetochemistry and single-crystal X-ray diffractometry. The UV-vis spectrum of complex 1 exhibits a strong peak at 510 nm, a characteristic absorption of a d-d transition of the square-planar four-coordinated Ni(II) center. Utilizing this feature, the stepwise formation of mono- and dinickel centers in solution can be monitored. Phosphodiesterase activity of a dinuclear Ni(II) system (complex 1), formed in situ by a 2 : 1 mixture of Ni(2+) ions and the ligand HL, was investigated using bis(4-nitrophenyl)phosphate (BNPP) as the substrate. The pH dependence of the BNPP cleavage in water-ethanol (1 : 1, v/v) reveals a bell-shaped pH-k(obs) profile with an optimum at about pH 8.3 which is parallel to the formation of the dinuclear species [Ni₂L(μ-OH)](2+), according to the increase of the peak at 510 nm in the UV-vis absorption spectrum . These studies reveal that the di-Ni(II) system shows the highest catalytic activity reported so far, with an acceleration rate 1.28 × 10⁷ times faster than the uncatalyzed reaction. The bridging hydroxyl group in [Ni₂L(μ-OH)](2+) is responsible for the hydrolysis reaction. The possible mechanism for the BNPP cleavage promoted by di-Ni(II) system is proposed on the basis of kinetic and spectral analyses. This study provides a less common example of the asymmetric phosphodiesterase model, which is like the active sites of most native metallohydrolases.

Carboxy and diphosphate ester hydrolysis promoted by di- or tri-nuclear zinc(II) complexes based on β-cyclodextrin

A new ligand (L), 6-mono-(2-(2-hydroxy-3-(hydroxymethyl)-5-methyl benzylamino)-ethylamino)-β-cyclodextrin, based on β-cyclodextrin derivatives with dinucleating units was synthesized and used to prepare a trimetallic bis-ligands zinc complex (Zn 3(L 2−) 2). The esterase activity of the complex was investigated by the hydrolysis of two carboxylic acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl acetate (NA), and a DNA model bis(4-nitrophenyl)phosphate (BNPP) as a phosphate ester. The catalytic rate for BNPC was very high, which was found to be a 5.63 × 10 3-fold rate enhancement over uncatalyzed hydrolysis and 1.62 × 10 2-fold rate enhancement over uncatalyzed hydrolysis for NA hydrolysis at pH = 7.0. For the catalytic hydrolysis of BNPP, the initial first-order rate constant of 0.1 mM catalyst was 5.85 × 10 −8 s −1 at pH = 8.50 and 35 °C, which is a 731-fold acceleration over uncatalyzed hydrolysis. The second rate constant ( k BNPP) was found to be 1.22 × 10 −3 M −1 s −1 at pH = 10.0. According to the potentiometric titration study, the zinc complex exists in a dinuclear single ligand coordinated mode and a trinuclear bis-ligands system at pH ≥ 7.0. The ester hydrolysis activity was attributed to the cooperative interaction of the two metal centers and the hydrophobic cavity of β-cyclodextrin with substrates.

Ester catalytic hydrolysis by a tridentate N,N′,N″-copper bridged cyclodextrin dimer

Abstract A new pyridine bridged cyclodextrin dimer mono-copper complex (CuL) was synthesized and characterized. The hydrolysis of carboxylic acid esters, bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl acetate (NA), and phosphate ester, a DNA model bis(4-nitrophenyl)phosphate (BNPP), promoted by CuL has been investigated. The resulting hydrolysis rate constants showed that CuL had a very high rate of catalysis for BNPC hydrolysis, yielding a 2.73 × 103-fold rate enhancement over uncatalyzed hydrolysis at pH 7.00, compared to only a 78.2-fold rate enhancement for NA hydrolysis. The initial first-order rate constant of catalytic hydrolysis for BNPP was 1.01 × 10−7 s−1 at pH 8.5, 35 °C and 0.1 mM catalyst concentration, about 1260-fold acceleration over uncatalyzed hydrolysis. The second rate constant kBNPP of BNPP hydrolysis promoted by CuL was found to be 5.94 × 10−4 M−1 s−1.

Synthesis of TiO2 Nanoparticles with Narrow Size Distribution and Their Evaluation in the Photocatalytic Oxidative Degradation of Bis(4-nitrophenyl) Phosphate

A new synthesis method of anatase titanium dioxide (TiO2) nanoparticles (NPs) of an average size of 2.1 ± 0.3 nm is reported. This is a fast, inexpensive, one-pot, and one-step method. It takes place under normal reaction conditions in dimethyl sulfoxide (DMSO) colloidal dispersions. The electronic absorption spectrum of these TiO2 colloidal particles shows an absorption band edge blue shift with respect to the macrocrystal due to the quantum confinement effect. After TiO2 NPs precipitation, the isolated powder changes its crystalline phase with the annealing process, up to 500 °C anatase is the unique phase, and at 1000 °C the precipitate is totally rutile. UV irradiated aqueous TiO2 NPs suspensions play an active role in the degradation of the bis-4-nitrophenyl phosphate (BNPP), as well as of its primary decomposition products, the 4-nitrophenyl phosphate (4-NPP) and the 4-nitrophenol (4-NP). As far as we now, the complete BNPP and 4-NPP photocatalytic or just catalytic degradation, using inorganic nano...

Activation of a PARACEST agent for MRI through selective outersphere interactions with phosphate diesters.

Ln(S-THP)(3+) complexes are paramagnetic chemical exchange saturation transfer (PARACEST) agents for magnetic resonance imaging (MRI; S-THP = (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane, Ln(III) = Ce(III), Eu(III), Yb(III)). CEST spectra at 11.7 T show that the PARACEST effect of these complexes is enhanced at neutral pH in buffered solutions containing 100 mM NaCl upon the addition of 1-2 equiv of diethylphosphate (DEP). CEST images of phantoms at 4.7 T confirm that DEP enhances the properties of Yb(S-THP)(3+) as a PARACEST MRI agent in buffered solutions at neutral pH and 100 mM NaCl. Studies using (1)H NMR, direct excitation Eu(III) luminescence spectroscopy, and UV-visible spectroscopy show that DEP is an outersphere ligand. Dissociation constants for [Ln(S-THP)(OH(2))](DEP) are 1.9 mM and 2.8 mM for Ln(III) = Yb(III) at pH 7.0 and Eu(III) at pH 7.4. Related ligands including phosphorothioic acid, O,O-diethylester, ethyl methylphosphonate, O-(4-nitrophenylphosphoryl)choline, and cyclic 3,5-adenosine monophosphate do not activate PARACEST. BNPP (bis(4-nitrophenyl phosphate) activates PARACEST of Ln(S-THP)(3+) (Ln(III) = Eu(III), Yb(III)), albeit less effectively than does DEP. These data show that binding through second coordination sphere interactions is selective for phosphate diesters with two terminal oxygens and two identical ester groups. A crystal structure of [Eu(S-THP)(OH(2))]((O(2)NPhO)(2)PO(2))(2)(CF(3)SO(3)) x 2 H(2)O x iPrOH has two outersphere BNPP anions that form hydrogen bonds to the alcohol groups of the macrocycle and the bound water ligand. This structure supports (1)H NMR spectroscopy studies showing that outersphere interactions of the phosphate diester with the alcohol protons modulate the rate of alcohol proton exchange to influence the PARACEST properties of the complex. Further, DEP interacts only with the nonionized form of the complex, Ln(S-THP)(OH(2))(3+) contributing to the pH dependence of the PARACEST effect.

Hydrolytic cleavage of DNA-model substrates promoted by polyoxovanadates

Hydrolysis of 4-nitrophenyl phosphate (NPP) and bis-4-nitrophenyl phosphate (BNPP), two commonly used DNA model substrates, was examined in vanadate solutions by means of (1)H, (31)P and (51)V NMR spectroscopy. The hydrolysis of the phosphoester bond in NPP at 50 degrees C and pH 5.0 proceeds with a rate constant of 1.74 x 10(-5) s(-1). The cleavage of the phosphoester bond in BNPP at 70 degrees C and pH 5.0 proceeds with a rate constant of 3.32 x 10(-6) s(-1), representing an acceleration of four orders of magnitude compared to the uncatalyzed cleavage. Inorganic phosphate and nitrophenol (NP) were the only products of hydrolysis. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of V(V) reduction to V(IV), indicating that the cleavage of the phosphoester bond is purely hydrolytic. The pH dependence of k(obs) revealed that the hydrolysis proceeds fastest in solutions of pH 5.5. Comparison of the rate profile with the concentration profile of polyoxovanadates shows a striking overlap of the k(obs) profile with the concentration of decavanadate (V(10)). Kinetic experiments at 37 degrees C using a fixed amount of NPP and increasing amounts of V(10) permitted the calculation of catalytic (k(c) = 5.67 x 10(-6) s(-1)) and formation constants for the NPP-V(10) complex (K(f) = 71.53 M(-1)). Variable temperature (31)P NMR spectra of a reaction mixture revealed broadening and shifting of the (31)P resonance upon addition of increasing amounts of decavanadate and upon increasing temperature, implying the dynamic exchange process between free and bound NPP at higher temperatures. The origin of the hydrolytic activity of V(10) is most likely due its high lability and its dissociation into smaller fragments which may allow the attachment of NPP and BNPP into the polyoxovanadate framework.

Binding and hydrolysis studies of antitumoural titanocene dichloride and Titanocene Y with phosphate diesters

The interaction of the antitumoural metallocene dihalides, titanocene dichloride (Cp(2)TiCl(2)) and Titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) chloride), with bis(4-nitrophenyl) phosphate (BNPP), which is a widely used model for the phosphate diester linkages in DNA, has been studied. Cp(2)TiCl(2) has been shown to promote the cleavage of the phosphate diester in weakly acidic solution. At pH 4, 37 degrees C, a 10(6)-fold rate acceleration over the uncatalysed reaction was observed under pseudo-first-order conditions, when freshly prepared solutions of Cp(2)TiCl(2) were applied. The activity of aged solutions dropped significantly due to the formation of insoluble precipitates of hydrolysed Ti species. The precipitates isolated from aged solutions were shown to act as moderately active, heterogeneous catalysts for BNPP cleavage. By contrast, no hydrolysis of the phosphate diester could be observed in the presence of Titanocene Y. Implications for the mode of action of the apoptosis-inducing metallocene dihalides are discussed.

Ester Hydrolysis by a Cyclodextrin Dimer Catalyst with a Tridentate N,N′,N′′‐Zinc Linking Group

A new beta-cyclodextrin dimer, 2,6-dimethylpyridine-bridged-bis(6-monoammonio-beta-cyclodextrin) (pyridyl BisCD, L), is synthesized. Its zinc complex (ZnL) is prepared, characterized, and applied as a catalyst for diester hydrolysis. The formation constant (log K(ML)=7.31+/-0.04) of the complex and deprotonation constant (pK(a1)=8.14+/-0.03, pK(a2)=9.24+/-0.01) of the coordinated water molecule were determined by a potentiometric pH titration at (25+/-0.1) degrees C, indicating a tridentate N,N',N''-zinc coordination. Hydrolysis kinetics of carboxylic acid esters were determined with bis(4-nitrophenyl)carbonate (BNPC) and 4-nitrophenyl acetate (NA) as the substrates. The resulting hydrolysis rate constants show that ZnL has a very high rate of catalysis for BNPC hydrolysis, yielding an 8.98x10(3)-fold rate enhancement over uncatalyzed hydrolysis at pH 7.00, compared to only a 71.76-fold rate enhancement for NA hydrolysis. Hydrolysis kinetics of phosphate esters catalyzed by ZnL are also investigated using bis(4-nitrophenyl)phosphate (BNPP) and disodium 4-nitrophenyl phosphate (NPP) as the substrates. The initial first-order rate constant of catalytic hydrolysis for BNPP was 1.29x10(-7) s(-1) at pH 8.5, 35 degrees C and 0.1 mM catalyst concentration, about 1600-fold acceleration over uncatalyzed hydrolysis. The pH dependence of the BNPP cleavage in aqueous buffer was shown as a sigmoidal curve with an inflection point around pH 8.25, which is nearly identical to the pK(a) value of the catalyst from the potentiometric titration. The k(BNPP) of BNPP hydrolysis promoted by ZnL is found to be 1.68x10(-3) M(-1) s(-1), higher than that of NPP, and comparatively higher than those promoted by its other tridentate N,N',N''-zinc analogues.

Highly efficient hydrolysis of phosphodiester by a copper(II)-chelated chitosan magnetic nanocarrier

A novel heterogeneous magnetic nanocatalyst was designed by conjugating copper(II) ions with chitosan-bound magnetic nanocarrier. Its hydrolytic activity for phosphodiesters was evaluated by investigating the hydrolysis of secondary ester bis-(nitrophenyl) phosphate (BNPP) in aqueous media. The maximum hydrolysis rate of BNPP was observed around the p K a of nanocatalyst which was found to be 12. The kinetic studies revealed that the binding affinity of BNPP to the copper(II)-chelated chitosan magnetic nanocarrier was low as compared to other catalyst systems. However, the rate constants ( k cat) and maximum reaction rate ( V max) were found to be higher with lower activation energy for the developed nanocatalyst. Moreover, the developed nanocatalyst was magnetically recoverable and also had good reusability.

X-ray structure and spectroscopic characterization of divalent dinuclear cobalt complexes containing carboxylate- and phosphodiester- auxiliary bridges

Two dinuclear spin-coupled divalent cobalt complexes, [Co 2(P1-O)(μ 2-OAc)](ClO 4) 2, ( 1) and [Co 2(P1-O)(μ 2-BNPP)](ClO 4) 2, ( 2) containing μ-1,3 acetate (OAc) and bis(4-nitrophenyl)phosphate (BNPP) auxiliary bridges, respectively, were synthesized by the reaction of a classic dinucleating ligand, P1–OH with cobalt(II) perchlorate in presence of acetic acid/bis(4-nitrophenyl)phosphate. They were characterized by single crystal X-ray diffraction, to show a trigonal bipyramidal geometry around each cobalt center and the intervening bridging atoms that are responsible for spin-transfer between the two divalent cobalt centers; the alkoxo oxygen donor occupies an equatorial position, and the auxiliary ligand oxygens (OAc/BNPP) occupy the axial positions. Solution state magnetic moment measurement together with UV–Vis/NIR spectra revealed a high-spin ground state ( S = 3/2) for Co(II) in these compounds. Complexes 1 and 2 show interesting 1H NMR spectral features of resonances with relatively narrower linewidths in conjunction with a sizable chemical shift dispersion of −5 and 265 ppm. Complex 2 containing the bis(4-nitrophenyl)phosphate auxiliary bridge showed narrower spectral window than complex 1 that has the acetate auxiliary bridge.

Copper(II) Complexes of N‐Methylated Derivatives of ortho‐ and meta‐Xylyl‐Bridged Bis(1,4,7‐triazacyclononane) Ligands: Synthesis, X‐ray Structure and Reactivity as Artificial Nucleases

AbstractTwo new binucleating ligands, 1,3‐bis[(4,7‐dimethyl‐1,4,7‐triazacyclononan‐1‐yl)methyl]benzene (Lmemx) and 1,2‐bis[(4,7‐dimethyl‐1,4,7‐triazacyclononan‐1‐yl)methyl]benzene(Lmeox), have been prepared from 1,4,7‐triazatricyclo[5.2.1.04,10]decane and applied in the synthesis of the corresponding copper(II) complexes, [Cu2(μ‐AcO)2(Lmemx)](ClO4)2·H2O (Cm) and [Cu2(η2‐AcO)2(Lmeox)](ClO4)2 (Co). The X‐ray crystal structures confirmed the dinuclear nature of the complexes, with pairs of copper(II) centres residing in distorted square‐pyramidal coordination environments. The nitrogen donors on the ligands occupy three facially disposed coordination sites about each copper(II) centre, with two acetate ligands completing the copper(II) coordination spheres. For Cm, both acetate groups bridge the two metal centres, whilst in Co each acetate chelates to a single metal centre. To assess their utility as simple nuclease mimics, the metal complexes were tested for their phosphate ester cleavage ability using the model phosphodiester, bis(4‐nitrophenyl)phosphate (BNPP). It was found that Co exhibited the fastest rate of BNPP hydrolysis (kobsd. = 2.4 × 10–5 s–1), this rate being five times faster than that of the CuII complex of the non‐methylated analogue. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Ester Hydrolysis by a Cyclodextrin Dimer Catalyst with a Metallophenanthroline Linking Group

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.

Pharmacological effects of a novel isosorbide-based butyrylcholinesterase inhibitor

Isosorbide-2-benzylcarbamate-5-benzoate, a novel butyrylcholinesterase inhibitor, shows interspecies variation in its inhibitory activity (IC 50 of 4.3 nM for human plasma butyrylcholinesterase, but 1.09 μM for mouse plasma butyrylcholinesterase). Stability studies revealed that this drug is resistant to hydrolysis by human plasma (no degradation in 1 h). However, it was found to undergo rapid degradation when incubated with mouse plasma or mouse liver homogenate, yielding benzyl carbamate and benzoic acid. The addition of the carboxylesterase inhibitor bis-(4-nitrophenyl) phosphate (BNPP) inhibited the degradation of the novel drug, indicating that it may be a substrate for both butyrylcholinesterase and carboxylesterase. The absence of carboxylesterase from human plasma explains the drug's stability in this medium. In vivo, pharmacodynamic studies on single doses of 1 mg/kg to naïve male C57BL/6 mice revealed maximal plasma butyrylcholinesterase inhibition 20 min after intraperitoneal administration (∼60% inhibition) and 1 h after administration by gavage (∼45% inhibition). While this plasma butyrylcholinesterase inhibition was short-lived, the drug also penetrated the blood–brain barrier resulting in a slight (10–15%) but persistent (≥72 h) reduction in brain butyrylcholinesterase activity.

1,4,7,10-Tetraazacyclododecane Metal Complexes as Potent Promoters of Phosphodiester Hydrolysis under Physiological Conditions

Previously reported mono- and dinuclear Zn(II), Cu(II), and Ni(II) complexes of 1,4,7,10-tetrazacyclododecane ([12]aneN4 or cyclen) with different heterocyclic spacers (triazine, pyridine) of various lengths (bi- and tripyridine) or an azacrown-pendant have been tested for the hydrolysis of bis(4-nitrophenyl)phosphate (BNPP) under physiological conditions (pH 7-9, 25 degrees C). All Zn(II) complexes promote the hydrolysis of BNPP under physiological conditions, while those of Cu(II) and Ni(II) do not have a significant effect on the hydrolysis reaction. The hydrolysis kinetics in buffered solutions (0.05 M Bis/Tris, TRIS, HEPES, or CHES, I=0.1 M, NaCl) at 25 degrees C were determined by the initial slope method (product conversion<5%). Comparison of the second-order pH-independent rate constants (kBNPP, M(-1) s(-1)) for the mononuclear complexes ZnL1, ZnL3, and ZnL6, which are 6.1x10 (-5), 5.1x10(-5), and 5.7x10(-5), respectively, indicate that the heterocyclic moiety improves the rate of hydrolysis up to six times over the parent Zn([12]aneN4) complex (kBNPP=1.1x10(-5) M(-1) s(-1)). The reactive species is the Zn(II)-OH- complex, in which the Zn(II)-bound OH- acts as a nucleophile. For dinuclear complexes Zn2L2, Zn2L4, and Zn2L5, the rate of reaction is defined by the degree of cooperation between the metal centers, which is determined by the spacer length. Zn2L2 and Zn2L4 possessing shorter spacers are able to hydrolyze BNPP 1 to 2 orders of magnitudes faster than Zn2L5. The second-order rate constants k of Zn2L4 and Zn2L2 at pH 7, 8, and 9 are significantly higher than those of previously reported related complexes. The high BNPP hydrolytic activity may be related to pi-stacking and hydrophobic interactions between the aromatic spacer moieties and the substrate. Complexes Zn2L4 and Zn2L2 show hydrolytic activity at pH 7 and 8, which allows for the hydrolysis of activated phosphate esters under physiological conditions.

Simplified Speciation and Improved Phosphodiesterolytic Activity of Hydroxo Complexes of Trivalent Lanthanides in Aqueous DMSO

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).

Copper(II) and Cobalt(II) Dinuclear Complexes with Oxime‐based Ligand as Catalysts for the Hydrolysis of Phosphate Diester

AbstractDinuclear copper(II) and cobalt(II) complexes were prepared and investigated with respect to their effects on hydrolysis of bis(4‐nitrophenyl) phosphate (BNPP). The kinetic data indicated that the mono‐deprotonated oxime group could act as an effective intramolecular nucleophile in the reaction. The higher reactivity of dinuclear copper(II) complex than that of its mononuclear analogues illustrated the probability of double Lewis acid activation by two metal centers in the former complex.

Specificity of Procaine and Ester Hydrolysis by Human, Minipig, and Rat Skin and Liver

The capacity of human, minipig, and rat skin and liver subcellular fractions to hydrolyze the anesthetic ester procaine was compared with carboxylesterase substrates 4-methylumbelliferyl-acetate, phenylvalerate, and para-nitrophenylacetate and the arylesterase substrate phenylacetate. Rates of procaine hydrolysis by minipig and human skin microsomal and cytosolic fractions were similar, with rat displaying higher activity. Loperamide inhibited procaine hydrolysis by human skin, suggesting involvement of human carboxylesterase hCE2. The esterase activity and inhibition profiles in the skin were similar for minipig and human, whereas rat had a higher capacity to metabolize esters and a different inhibition profile. Minipig and human liver and skin esterase activity was inhibited principally by paraoxon and bis-nitrophenyl phosphate, classical carboxylesterase inhibitors. Rat skin and liver esterase activity was inhibited additionally by phenylmethylsulfonyl fluoride and the arylesterase inhibitor mercuric chloride, indicating a different esterase profile. These results have highlighted the potential of skin to hydrolyze procaine following topical application, which possibly limits its pharmacological effect. Skin from minipig used as an animal model for assessing transdermal drug preparations had similar capacity to hydrolyze esters to human skin.