Artificial Phosphodiesterases Research Articles

Artificial phosphatase upon premicellar nanoarchitectonics of lanthanum complexes with long-chained imidazole derivatives

Four novel long chain-containing tridentate imidazole derivatives (Ln, n = 1, 2, 3, 4) were synthesized for in situ formation of mononuclear lanthanum(III) complexes as artificial phosphodiesterases. These in-situ formed La(III) complexes (named LaLn) were used to catalyze the transesterification of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP), a classic RNA model. Critical aggregation concentrations (CAC) were determined for the as-prepared tridentate imidazole derivatives as ligands and corresponding mixtures of equivalent ligand and La3+ ion with a mole rate of 1:1. It denotes that the introduction of La3+ ion increases the CAC values of imidazole derivatives by about 2 to 3 folds. Foaming test shows that the foam height is positively correlated with the length of hydrophobic chain. Transesterification of HPNP mediated by LaLn nanoarchitectonics indicates that the introducing of hydrophobic chain benefits rate enhancement, showing excess three orders of magnitude acceleration under physiological conditions (pH 7.0, 25 °C). Moreover, catalytic reactivities of these La(III) complexes increased along with the increase in chain length: LaL1 < LaL2 < LaL3 < LaL4, suggesting a positive correlation to hydrophobic chain length.

Upper Rim Bifunctional cone-Calix[4]arenes Based on a Ligated Metal Ion and a Guanidinium Unit as DNAase and RNAase Mimics.

The catalytic activity of an artificial phosphodiesterase that combines a ligated metal ion (Cu(II), Zn(II)) with a guanidinium unit connected by a 1,2-vicinal calix[4]arene spacer was investigated in the transesterification of RNA models HPNP and four diribonucleoside 3',5'-monophosphates. Comparison with previous data related to the 1,3-distal regioisomeric metal complexes confirms the superiority of the Cu(II) complexes over the Zn(II) analogs and shows that in the reactions of HPNP, GpU, and UpU, the catalytic efficiency depends very little on whether the substitution pattern is 1,2-vicinal or 1,3-distal. On the other hand, CpA turned out to be a good substrate for the Cu(II) complex of the 1,2-vicinal catalyst and a bad substrate for the corresponding 1,3-distal regioisomer, whereas the opposite holds for GpA. Extension of the investigation to the cleavage of the DNA model BNPP showed that both Zn(II) and Cu(II) complexes exhibit good catalytic efficiency, with a superiority of the 1,2-vicinal catalyst in both cases. The data reported in this work show that rate accelerations over background for the best catalyst-substrate combinations at 0.5 mM catalyst concentration are 3.6 × 10(5)-fold for HPNP, 1.1 × 10(6)-fold for BNPP, and range from 1.3 × 10(6)- to 1.3 × 10(7)-fold for diribonucleoside monophosphates.

Phosphodiester cleavage by trivalent lanthanides in the presence of native cyclodextrins

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 2mM 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.

ChemInform Abstract: The Guanidinium Unit in the Catalysis of Phosphoryl Transfer Reactions: From Molecular Spacers to Nanostructured Supports

AbstractReview: [64 refs.

The guanidinium unit in the catalysis of phosphoryl transfer reactions: from molecular spacers to nanostructured supports.

Examples of guanidinium-based artificial phosphodiesterases are illustrated in this review article. A wide set of collected catalytic systems are presented, from the early examples to the most recent developments of the use of this unit in the design of supramolecular catalysts. Special attention is dedicated to illustrate the operating catalytic mechanism and the role of guanidine/ium units in the catalysis. One or more of these units can act by themselves or in conjunction with other active units. The analogy with the mechanism of enzymatic systems is presented and discussed. In the last part of this overview, recent examples of guanidinophosphodiesterases based on nanostructured supports are reported, namely gold-monolayer-protected clusters and polymer brushes grafted to silica nanoparticles. The issue of the dependence of the catalytic performance on the preorganization of the spacer is tackled and discussed in terms of effective molarity, a parameter that can be taken as a quantitative measurement of this preorganization for both conventional molecular linker and nanosized supports.

Guanidine-based polymer brushes grafted onto silica nanoparticles as efficient artificial phosphodiesterases.

Polymer brushes grafted to the surface of silica nanoparticles were fabricated by atom-transfer radical polymerization (ATRP) and investigated as catalysts in the cleavage of phosphodiesters. The surfaces of silica nanoparticles were functionalized with an ATRP initiator. Surface-initiated ATRP reactions, in varying proportions, of a methacrylate moiety functionalized with a phenylguanidine moiety and an inert hydrophilic methacrylate species afforded hybrid nanoparticles that were characterized with potentiometric titrations, thermogravimetric analysis, and SEM. The activity of the hybrid nanoparticles was tested in the transesterification of the RNA model compound 2-hydroxypropyl para-nitrophenylphosphate (HPNP) and diribonucleoside monophosphates. A high catalytic efficiency and a remarkable effective molarity, thus overcoming the effective molarities previously observed for comparable systems, indicate the existence of an effective cooperation of the guanidine/guanidinium units and a high level of preorganization in the nanostructure. The investigated system also exhibits a marked and unprecedented selectivity for the diribonucleoside sequence CpA. The results presented open up the way for a novel and straightforward strategy for the preparation of supramolecular catalysts.

Guanidine based self-assembled monolayers on Au nanoparticles as artificial phosphodiesterases

Gold nanoparticles passivated with a catalytic monolayer based on guanidine exhibit high cooperativity and efficiency in the cleavage of phosphodiesters.

Guanidine–Guanidinium Cooperation in Bifunctional Artificial Phosphodiesterases Based on Diphenylmethane Spacers; gem-Dialkyl Effect on Catalytic Efficiency

Diphenylmethane derivatives 1-3, decorated with two guanidine units, are effective catalysts of HPNP transesterification. Substitution of the methylene group of the parent diphenylmethane spacer with cyclohexylidene and adamantylidene moieties enhances catalytic efficency, with gem-dialkyl effect accelerations of 4.5 and 9.1, respectively. Activation parameters and DFT calculations of the rotational barriers around the C-Ar bonds indicate that a major contribution to the driving force for enhanced catalysis is entropic in nature.

Upper Rim Guanidinocalix[4]arenes as Artificial Phosphodiesterases

Calix[4]arene derivatives, blocked in the cone conformation and functionalized with two to four guanidinium units at the upper rim were synthesized and investigated as catalysts in the cleavage of the RNA model compound 2-hydroxypropyl p-nitrophenyl phosphate. When compared with the behavior of a monofunctional model compound, the catalytic superiority of the calix[4]arene derivatives points to a high level of cooperation between catalytic groups. Combination of acidity measurements with the pH dependence of catalytic rates unequivocally shows that a necessary requisite for effective catalysis is the simultaneous presence, on the same molecular framework, of a neutral guanidine acting as a general base and a protonated guanidine acting as an electrophilic activator. The additional guanidinium (guanidine) group in the diprotonated (monoprotonated) trifunctional calix[4]arene acts as a more or less innocent spectator. This is not the case with the tetrasubstituted calix[4]arene, whose mono-, di-, and triprotonated forms are slightly less effective than the corresponding di- and triguanidinocalix[4]arene derivatives, most likely on account of a steric interference with HPNP caused by overcrowding.

General Base–Guanidinium Cooperation in Bifunctional Artificial Phosphodiesterases

Artificial phosphodiesterases that combine a guanidinium unit with a general base connected by a m-xylylene linker catalyze the transesterification of the RNA model compound 2-hydroxypropyl p-nitrophenyl phosphate (HPNP). The bifunctional catalysts presented in this work show varying extents of cooperation between catalytic units and a rate enhancement of 4 × 10(4) in the most favorable case.

Metal ion induced allosteric transition in the catalytic activity of an artificial phosphodiesterase

An artificial phosphodiesterase () bearing two types of metal binding sites, a catalytic site and a regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal concentration. The rate constants for the hydrolysis reaction of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) and RNA dimer (ApA) with and without an effector metal ion were evaluated; the k(obs) value of HPNP hydrolysis for .(Zn(2+))(3) (2.0 x 10(-4) s(-1)) is 3.3 times larger than that for .(Zn(2+))(2). In the case of and Cu(2+), a 19.4 times larger k(obs) value was obtained for .(Cu(2+))(3) (1.2 x 10(-3) s(-1)) against .(Cu(2+))(2). The increase in the catalytic activity is ascribed to the allosteric conformational transition of induced by the coordination of effector metal ion to the Bpy moiety. A detailed investigation revealed that a conformational change of induced by the third M(2+) complexation enhances the rate of hydrolysis rather than a change in the substrate affinity.

Phosphodiesterase Activity of CvfA Is Required for Virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.

Metal ion induced allosteric transition in the catalytic activity of an artificial phosphodiesterase.

An artificial phosphodiesterase (1) bearing two kinds of metal binding sites, a catalytic site and a regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal concentration.

An allosteric synthetic catalyst: metal ions tune the activity of an artificial phosphodiesterase.

A trinuclear metal complex of general formula (L-H)M3(Mf)2 represents the first allosteric low molecular weight catalyst. L is a polyaza ligand having a tetradentate and two bidentate metal binding sites, Ms is a "structural" (allosteric) metal, and Mf are functional (catalytic) metals which interact with a substrate. In mononuclear [(L-H)Ms]+ complexes [(L-H)Cu(MeOH)]ClO4 (1a). [(L-H)Cu]NO3 x 2H2O (1b), [(L-H)Ni]ClO4 x 4H2O (2), and [(L-H)Pd]ClO4 x 2H2O (3), prepared from L and M2+ salts, the metal is strongly bound by an in-plane N4-coordination (confirmed by X-ray crystal structure determination of la). Formation of trinuclear complexes [(L-H)MsCu2]5+, with two functional Cu2+ ions coordinated to the bidentate sites of L, was evidenced in solution by photometric titration and by isolation of [(L-H)Cu3][PO4][ClO4]2 x 9H2O (4). The trinuclear complexes catalyze the cleavage of RNA-analogue 2-(hydroxypropyl)-p-nitrophenyl phosphate (HPNP), an activated phosphodiester. From a kinetic analysis of the cleavage rate at various HPNP concentrations, parameters KHPNP (the equilibrium constant for binding of HPNP to [(L-H)MsCu2]5+ and kcat (first-order rate constant for cleavage of HPNP when bound to the catalyst) were derived: KM= 170 (Ms= Cu2+), 340 (Ms = Ni2+), 2,600 (Ms = Pd2+) M(-1), kcat = 17 x 10(-3) (Ms= Cu2+) 3.1 x 10(-3) (Ms=Ni2+), 0.22 x 10(-3) (Ms = Pd2+) s(-1). Obviously, the nature of the allosteric metal ion Ms strongly influences both substrate affinity and reactivity of the catalyst [(L-H)MsCu2]5+. Our interpretation of this observation is that subtle differences in the ionic radius of Ms and in its tendency to distort the N4-Ms coordination plane have a significant influence on the conformation of the catalyst (i.e., preorganization of functional Cu2+ ions) and thus on catalytic activity.

Nucleobase-containing metallated polymeric resins as artificial phosphodiesterases: kinetics of hydrolysis, pH dependence, and catalyst recycling.

Two metallated polymeric resins that contain the nucleobase adenine have been investigated for their ability to catalyze the hydrolysis of model phosphodiester substrates. These resins behave in a catalytic manner and display classical Michaelis-Menten kinetics. Consequently, parameters such as kobs, Km, Vmax, and kcat were determined for the two resins for both of the substrates. The most attractive feature of our nucleolytic system is its facile reusability that permits catalysis of multiple hydrolytic reactions, after resin recovery and a simple washing step, without significantly compromising the reaction rate. Involvement of a hydrolytic mechanism for phosphodiester cleavage is proposed on the basis of pH versus hydrolytic rate profiles for the two resins. We have also been able to demonstrate temperature-dependence, solvent effects, and inhibitory nature of vanadate ions on the observed rate of hydrolytic reaction aided by the resins. In conclusion, metallated nucleobase resins represent a new class of nucleolytic reagents and these systems have the potential to be further developed for multifarious applications in chemical biology.

Using guanidinium groups for the recognition of RNA and as catalysts for the hydrolysis of RNA

The guanidinium functional group is commonly used in nature to recognize and bind anions through ion pairing and hydrogen bonding. Specific hydrogen-bonding patterns can be found in crystal structures of simple guanidinium salts. Analysis of these simple salts reveals a variety of features which are found in natural systems. These features have been applied to a series of artificial phosphodiesterases for RNA. These receptors incorporate guanidinium groups positioned to mimic the hydrogen-bonding patterns found in simple guanidinium salts and natural enzymes. This paper outlines general guanidinium hydrogen-bonding patterns. Next, the complexation of phosphodiesters with a series of artificial receptors are analyzed in terms of counterions, solvent mixtures, and cavity flexibility. In addition, strategies to enhance catalysis through a p K a analysis of phosphoranes are addressed. Next, we describe how our findings were incorporated into second generation receptors/catalysts. Finally, our future work is discussed.

Dinucleosidetriphosphatase from rat brain

1. 1. Rat brain p 1, p 3-bis(5′-adenosyl)-triphosphate adenylohydrolase (dinucleosidetriphos-phatase, EC 3.6.1.29) was purified 1000-fold. 2. 2. The enzyme hydrolyzed diadenosine and diguanosine triphosphates ( K m values 14 and 40μM, and relative V 100 and 40, respectively) to the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was hydrolyzed at a residual rate. Diadenosine di and tetraphosphates, NAD +, and artificial phosphodiesterase substrates were not hydrolyzed. 3. 3. Bivalent cations [Mg(II), Mn(II) or Ca(II)] were required for activity, but Zn(II) was a competitive inhibitor ( K i = 5 μM). The optimum pH value was about 7.5. 4. 4. A molecular mass of 34 kdalton (gel filtration) and an isoelectric point of 5.5 (chromatofocusing) were found.