- Hydrolysis of 2'- and 3'-C-Methyluridine 2',3'-Cyclic Monophosphates and Interconversion and Dephosphorylation of the Resulting 2'- and 3'-Monophosphates: Comparision with the Reactions of Uridine Monophosphates
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2',3'-Cyclic monophosphates of 2'- and 3'-C-methyluridines have been prepared and shown to hydrolyze to a mixture of the corresponding 2'- and 3'-monophosphates.The predominant product isomer is the one having the tertiary hydroxyl group phosphorylated, but on longer treatment a phosphate migration from the tertiary to secondary hydroxyl function takes place.Hydrolytic dephosphorylation competes with the phosphate migration, the tertiary hydroxyl group being dephosphorylated 1 order of magnitude faster than the secondary one.Kinetics of the partial reactions have been described and compared to the data obtained with uridine 2',3'-cyclic monophosphate.The tertiary monophosphate has been shown to be exceptionally susceptible to nucleophilic attack of the neighboring hydroxyl group.
- Mikhailov, Sergey N.,Oivanen, Mikko,Oksman, Pentti,Loennberg, Harri
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- Ribonuclease Mimic: Zn2+ Promoted Cleavage of C8-Histamino-r(UpA) proceeds through 2',3'-cUMP as Intermediate
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The designed ribodinucleotide 1 containing a proximal imidazole, in the presence of ZnCl2, undergoes self hydrolysis faster than unmodified r(UpA); like its enzymatic counterpart, this reaction goes through the same steps of initial transesterification to 2',3'-cUMP followed by hydrolysis to 2'- or 3'-UMP.
- Prakash, Thazha P.,Ganesh, Krishna N.
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- Cleavage and isomerization of UpU promoted by dinuclear metal ion complexes
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The catalysis of phosphoryl transfer by metal ions has been intensively studied in both biological and artificial systems, but the status of the transient pentacoordinate phosphoryl species (as transition state or intermediate) is the subject of considerable debate. We report that dinuclear metal ion complexes that incorporate second sphere hydrogen bond donors not only promote the cleavage of RNA fragments just as efficiently as the activated analogue HPNPP but also provide the first examples of metal ion catalyzed phosphate diester isomerization close to neutral pH. This observation implies that the reaction catalyzed by these complexes involves the formation of a phosphorane intermediate that is sufficiently long-lived to pseudorotate. Copyright
- Linjalahti, Heidi,Feng, Guoqiang,Mareque-Rivas, Juan C.,Mikkola, Satu,Williams, Nicholas H.
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- Reactivity of a 2'-thio nucleotide analog
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The chemical reactivity of ribonucleotide analog 2'-deoxy-2'-thiouridine 3'-(p-nitrophenyl phosphate) (1), in which the 2' hydroxyl is replaced with a 2'-thiol group, has been characterized. The major reaction pathway for 1, as monitored by 31P NMR spectroscopy, is transphosphorylation to afford 2',3'-cyclic phosphorothioate 3, followed by hydrolysis of 3 to produce 2'-deoxy-2'-thiouridine 2'-phosphorothioate (4). Thus, the reaction pathway of 1 is similar to that of the hydrolysis of ribonucleotides, yet there are significant differences. The pH-rate profile for transphosphorylation of 1 was determined by monitoring the formation of p-nitrophenol or p-nitrophenolate by UV-visible spectroscopy. Analysis of the profile reveals the attacking nucleophile to be thiolate, and the pK(a) of the 2'-thiol was determined to be 8.3 ± 0.1. At pH 7.4, the thiol-containing ribonucleotide analog 1 is hydrolyzed at an observed rate 27-fold slower than its 2'-hydroxyl counterpart. These results indicate that the rate of thiolate attack on the adjacent phosphodiester bond is 107-fold slower than that of the corresponding alkoxide. Thiolate nucleophiles, therefore, are remarkably reticent toward attack at electrophilic phosphate centers. In addition to providing new information about the reactivity of phosphodiester bonds, our studies highlight the potential of 2'-thiol-containing nucleotides for the study of an array of RNA processes, especially those in which the 2'-substituent plays a critical role.
- Dantzman, Cathy L.,Kiessling, Laura L.
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- Substrate specificity of an active dinuclear Zn(II) catalyst for cleavage of RNA analogues and a dinucleoside
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The cleavage of the diribonucleoside UpU (uridylyl-3′-5′- uridine) to form uridine and uridine (2′,3′)-cyclic phosphate catalyzed by the dinuclear Zn(II) complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)- 2-hydroxypropane (Zn2(1)(H2O)) has been studied at pH 7-10 and 25 °C. The kinetic data are consistent with the accumulation of a complex between catalyst and substrate and were analyzed to give values of kc (S-1), Kd (M), and kc/K d (M-1 s-1) for the Zn2(1)(H 2O)-catalyzed reaction. The pH rate profile of values for log k C/Kd for Zn2(1)(H2O)-catalyzed cleavage of UpU shows the same downward break centered at pH 7.8 as was observed in studies of catalysis of cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) and uridine-3′-4-nitrophenyl phosphate (UpPNP). At low pH, where the rate acceleration for the catalyzed reaction is largest, the stabilizing interaction between Zn2(1)(H2O) and the bound transition states is 9.3, 7.2, and 9.6 kcal/mol for the catalyzed reactions of UpU, UpPNP, and HpPNP, respectively. The larger transition-state stabilization for Zn 2(1)(H2O)-catalyzed cleavage of UpU (9.3 kcal/mol) compared with UpPNP (7.2 kcal/mol) provides evidence that the transition state for the former reaction is stabilized by interactions between the catalyst and the C-5′-oxyanion of the basic alkoxy leaving group.
- O'Donoghue, Annmarie,Pyun, Sang Yong,Yang, Meng-Yin,Morrow, Janet R.,Richard, John P.
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- Buffer catalyzed cleavage of uridylyl-3′,5′-uridine in aqueous DMSO: Comparison to its activated analog, 2-hydroxypropyl 4-nitrophenyl phosphate
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Buffer catalysis of the cleavage and isomerization of uridylyl-3′,5′-uridine (UpU) has been studied over a wide pH range in 80% aq. DMSO. The diminished hydroxide ion concentration in this solvent system made catalysis by amine buffers (morpholine, 4-hydroxypiperidine and piperidine) visible even at relatively low buffer concentrations (10-200 mmol L-1). The observed catalysis was, however, much weaker than what has been previously reported for the activated RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) in the same solvent system. In the case of morpholine, contribution of both the acidic and the basic buffer constituent was significant, whereas with 4-hydroxypiperidine and piperidine participation of the acidic constituent could not be established unambiguously. The results underline the importance of using realistic model compounds, along with activated ones, in the study of the general acid/base catalysis of RNA cleavage.
- Lain,L?nnberg,L?nnberg
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- Hydrolytic dethiophosphorylation and desulfurization of the monothioate analogues of uridine monophosphates under acidic conditions
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The hydrolytic reactions of uridine 2′-, 3′- and 5′-phosphoromonothioates (2′-, 3′- and 5′-UMPS) under acidic and neutral conditions have been followed by HPLC. Under slightly acidic conditions (pH 2-5), only pH-independent dethiophosphorylation to uridine takes place. This reaction is 200- to 300-fold as fast as dephosphorylation of the corresponding uridine monophosphates (UMP), presumably due to higher stability of the thiometaphosphate monoanion compared to metaphosphate anion. At pH > 5, i.e. at pH > pKa2 of the thiophosphate moiety, the dethiophosphorylation is retarded with increasing basicity of the solution. At pH 1, acid-catalysed desulfurization of 2′- and 3′-UMPS to an isomeric mixture of 2′/3′-UMP competes with their dethiophosphorylation. This reaction is suggested to proceed by a nucleophilic attack of the neighbouring hydroxy group on phosphorus. No such reaction occurs with 5′-UMPS. In contrast to 2′- and 3′-UMP, no sign of interconversion of 2′- and 3′-UMPS is detected.
- Ora, Mikko,Oivanen, Mikko,Loennberg, Harri
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- Tethered dinuclear europium(III) macrocyclic catalysts for the cleavage of RNA
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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 ( 7F0 → 5D0) 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 (NaNO 3) 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°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.
- Nwe, Kido,Andolina, Christopher M.,Morrow, Janet R.
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p. 14861 - 14871
(2009/02/08)
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- Dinuclear Zn2+ complexes in the hydrolysis of the phosphodiester linkage in a diribonucleoside monophosphate diester.
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Dizinc complexes that were formed from 2:1 mixtures of Zn(NO3)2 and dinucleating ligands TPHP (1), TPmX (2) or TPpX (3) in aqueous solutions efficiently hydrolyzed diribonucleoside monophosphate diesters (NpN) under mild conditions. The dinucleating ligand affected the structure of the aquo-hydroxo-dizinc core, resulting in different characteristics in the catalytic activities towards NpN cleavage. The pH-rate profile of ApA cleavage in the presence of (Zn2+)(2)-1 was sigmoidal, whereas those of (Zn2+)(2)-2 and (Zn2+)(2)-3 were bell-shaped. The pH titration study indicated that (Zn2+)(2)-1 dissociates only one aquo proton (up to pH 12), whereas (Zn2+)(2)-2 dissociates three aquo protons (up to pH 10.7). The observed differences in the pH-rate profile are attributable to the various distributions of the monohydroxo-dizinc species, which are responsible for NpN cleavage. As compared to that using (Zn2+)(2)-1, the NpN cleavage using (Zn2+)(2)-2 showed a greater rate constant, with a higher product ratio of 3'-NMP/2'-NMP. The saturation behaviors of the rate, with regard to the concentration of NpN, were analyzed by Michaelis-Menten type kinetics. Although the binding of (Zn2+)(2)-2 to ApA was weaker than that of (Zn2+)(2)-1, (Zn2+)(2)-2 showed a greater kcat value than (Zn2+)(2)-1, resulting in higher ApA cleavage activity of the former.
- Yashiro, Morio,Kaneiwa, Hideki,Onaka, Kenichi,Komiyama, Makoto
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p. 605 - 610
(2007/10/03)
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- Phosphodiester Cleavage of Ribonucleoside Monophosphates and Polyribonucleotides by Homo- and Heterodinuclear Metal Complexes of a Cyclohexane-Based Polyamino-Polyol Ligand
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The ability of the dinuclear complexes of tdci [1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol] to promote the cleavage of the phosphodiester bonds of nucleoside 2′,3′-cyclic monophosphates, dinucleoside monophosphates and polyribonucleotides has been studied. The homodinuclear copper(II) and zinc(II) complexes efficiently promote the hydrolysis of cyclic nucleotides. The second-order rate constant (k2≈0.44M-1S-1) estimated for the cleavage of 2′,3′-cAMP induced by dinuclear copper(II) complexes is about 107 times greater than that for the hydroxide-ion-catalysed reaction. The complex selectively cleaves the 2′O-P bond of 2′,3′-cUMP and forms the 3′-product in 91% yield. An equimolar mixture of copper(II), zinc(II) and tdci proved to be more efficient than either of the binary systems: a 7-20-fold rate enhancement was observed for the cleavage of 2′,3′-cNMP substrates. The half-life for the hydrolysis of 2′,3′-cAMP decreased from 300 days to five minutes at 25°C when the concentration of each of the three components was 2.5mM. In contrast to the copper(II) or zinc(II) complexes of tdci, the heterodinuclear species promoted the hydrolysis of several dinucleoside monophosphates. For two ApA isomers, cleavage of the 3′,5′-bond was about 6.5 times faster than cleavage of the 2′,5′-bond. On the basis of the kinetic data, a trifunctional mechanism is suggested for the heterodinuclear-complex-promoted cleavage of the phosphodiester bond. Double Lewis acid activation occurs when the metal ions bind to the phosphate oxygen atoms. In particular, a metal-bound hydroxide ion serves as a general base or a nucleophilic catalyst, and, presumably, a zinc(II)-bound aqua ligand behaves as a general acid and facilitates the departure of the leaving alkoxide group. The effect of the complexes on the hydrolysis of poly(U), poly(A) and type III native RNA was also investigated, and, for the first time, kinetic data on the cleavage of the phosphodiester bonds of polyribonucleotides by a dinuclear complex was obtained.
- Jancso, Attila,Mikkola, Satu,Loennberg, Harri,Hegetschweiler, Kaspar,Gajda, Tamas
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p. 5404 - 5415
(2007/10/03)
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- The pKa of the internucleotidic 2′-hydroxyl group in diribonucleoside (3′→5′) monophosphates
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Ionization of the internucleotidic 2′-hydroxyl group in RNA facilitates transesterification reactions in Group I and II introns (splicing), hammerhead and hairpin ribozymes, self-cleavage in lariatRNA, and leadzymes and tRNA processing by RNase P RNA, as well as in some RNA cleavage reactions promoted by ribonucleases. Earlier, the pKa of 2′-OH in mono- and diribonucleoside (3′-5′) monophosphates had been measured under various nonuniform conditions, which make their comparison difficult. This work overcomes this limitation by measuring the pKa values for internucleotidic 2′-OH of eight different diribonucleoside (3′-5′) monophosphates under a set of uniform noninvasive conditions by 1H NMR. Thus the pKa is 12.31 (±0.02) for ApG and 12.41 (±0.04) for ApA, 12.73 (±0.04) for GpG and 12.71 (±0.08) for GpA, 12.77 (±0.03) for CpG and 12.88 (±0.02) for CpA, and 12.76 (±0.03) for UpG and 12.70 (±0.03) for UpA. By comparing the pKas of the respective 2′-OH of monomeric nucleoside 3′-ethyl phosphates with that of internucleotidic 2′-OH in corresponding diribonucleoside (3′→5′) monophosphates, it has been confirmed that the aglycons have no significant effect on the pKa values of their 2′-OH under our measurement condition, except for the internucleotidic 2′-OH of 9-adeninyl nucleotide at the 5′-end (ApA and ApG), which is more acidic by 0.3-0.4 pKα units.
- Acharya,Foeldesi,Chattopadhyaya
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p. 1906 - 1910
(2007/10/03)
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- Kinetic isotope effects and stereochemical studies on a ribonuclease model: Hydrolysis reactions of uridine 3'-nitrophenyl phosphate
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The reactions of a ribonuclease model substrate, the compound uridine-3'-p-nitrophenyl phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis. The cyclization of this compound is subject to catalysis by general base (by imidazole buffer), specific base (by carbonate buffer), and by acid. All three reactions proceed by the same mechanistic sequence, via cyclization to cUMP, which is stable under basic conditions but which is rapidly hydrolyzed to a mixture of 2'- and 3'-UMP under acid conditions. The isotope effects indicate that the specific base-catalyzed reaction exhibits an earlier transition state with respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction. Stereochemical analysis indicates that both of the base-catalyzed reactions proceed with the same stereochemical outcome. It is concluded that the difference in the nucleophile in the two base-catalyzed reactions results in a difference in the transition state structure but both reactions are most likely concerted, with no phosphorane intermediate. The 15N isotope effects were also measured for the reaction of the substrate with ribonuclease A. The results indicate that considerably less negative charge develops on the leaving group in the transition state than for the general base-catalyzed reaction in solution. (C) 2000 Academic Press.
- Hengge,Bruzik,Tobin,Cleland,Tsai
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p. 119 - 133
(2007/10/03)
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- Preparation and cleavage reactions of 3′-thiouridylyl-(3′→5′)-uridine
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3′-Thiouridylyl-(3′→5′)-uridine [(Us)pU] 3 is prepared by coupling together the disulfide 14 and the 5′-H-phosphonate 18, and then removing the protecting groups. (Us)pU 3 readily undergoes cleavage in 0.05 mol dm-3 sodium glycinate buffer (pH 10.06) at 50 °C to give, in the first instance, uridine 4 and 3′-thiouridine 2′,3′-cyclic phosphorothioate 21; in glacial acetic acid at 30 °C, it rapidly undergoes cleavage in essentially the same way. The behaviour of (Us)pU 3 is compared with that of uridylyl-(3′→5′)-uridine (UPU) 1a under the same basic and acidic reaction conditions. (Us)pU 3 and 3′-thiouridine 2′,3′-cyclic phosphorothioate 21 are both substrates for ribonuclease A; (Us)pU 3 is a substrate for Crotalus adamanteus snake venom phosphodiesterase but not for calf spleen phosphodiesterase.
- Liu, Xiaohai,Reese, Colin B.
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p. 2227 - 2236
(2007/10/03)
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- Rapid and highly base selective RNA cleavage by a dinuclear Cu(II) complex
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A bis-Cu(II) complex based on a covalently linked terpyridine and bipyridine ligand system is shown to rapidly cleave bis-ribonucleotides with remarkable selectivity for adenine bases.
- Liu, Shanghao,Hamilton, Andrew D.
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p. 587 - 588
(2007/10/03)
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- Rapid and highly selective cleavage of ribonucleoside 2',3'-cyclic monophosphates by dinuclear Cu(II) complexes
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Two characteristics that never before appeared together are combined in complexes 1 and 2, which show high activity and high selectivity in the hydrolysis of cyclic nucleoside 2',3'-monophosphates as model compounds for RNA. In the case of 1 the regioselectivity is exceptional, and, in the case of 2, the base selectivity.
- Liu,Luo,Hamilton
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p. 2678 - 2680
(2007/10/03)
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- Kinetics and Mechanism of Facile and Selective Dephosphorylation of 2′-Phosphorylated and 2′-Thiophosphorylated Dinucleotides: Neighboring 3′-5′ Phoshodiester Promotes 2′-Dephosphorylation
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2′-Phosphorylated and 2′-thiophosphorylated dinucleotides U(2′-p)pU (1) and U(2′-ps)pU (2) were found to undergo facile 2′-specific dephosphorylation at 90°C in neutral aqueous solution to give UpU, and the first-order rate constants of these reactions were determined by HPLC. Particularly, U(2′-ps)pU (2, k = 1.38 ± 0.4 × 10-3 s-1, tcomp = 1 h) was cleanly dephosphorylated ca. 100 times more rapidly than U(2′-p)pU (1, k = 1.41 ± 0.05 × 10-5 s-1, tcomp = 72 h). Dephosphorylations of 1 and 2 were faster than those of thymidine 3′-phosphate (8) and thymidine 3′-thiophosphate (9), respectively. The kinetic data observed were independent of the 2′- or 3′-position of the phosphate group and the kind of base moiety. The neighboring 3′-5′ phosphodiester function most probably promotes the 2′-dephosphorylation efficiently. A branched trimer, U(2′-pU)pU (3), and related compounds having a substituent on the 2′-phosphoryl group, such as U(2′-pp-biotin)pU (4) and U(2′-ps-bimane)pU (5), were rather resistant to hydrolysis. The addition of divalent metal ions (Mg2+, Mn2+, Zn2+, Ca2+, Co2+, and Cd2+) remarkably decreased the rate of 2′-de(thio)phosphorylation of 1 or 2. Among these metal ions, Zn2+ most significantly inhibited the dephosphorylation. On the contrary, trivalent metal ions considerably accelerated the 2′-de(thio)phosphorylation of 1 or 2. The mechanism of 2′-dephosphorylation in the presence and absence of various metal ions is also discussed.
- Tsuruoka, Hiroyuki,Shohda, Koh-Ichiroh,Wada, Takeshi,Sekine, Mitsuo
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p. 2813 - 2822
(2007/10/03)
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- Synthesis and properties of diuridine phosphate analogues containing thio and amino modifications
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Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10-6 s-1 at pH 6 to 3200 x 10-6 s-1 at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10-6 s-1 at pH 5 to 140 x 10-6 s-1 at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10-6 s-1 and 138 x 10-6 s-1. The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10-6 s-1 at pH 11.5 to 11 685 x 10-6 s-1 at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg2+,Zn2+, and Cd2+ ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of κ(cat).
- Thomson, James B.,Patel, Bhisma K.,Jimenez, Victor,Eckart, Klaus,Eckstein, Fritz
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p. 6273 - 6281
(2007/10/03)
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- Uridylyl-(3′ → 5′)-(5′-thiouridine). An exceptionally base-labile diribonucleoside phosphate analogue
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Like uridylyl-(3′→5′)-uridine (UpU), uridylyl-(3′→5′)-(5′-thiouridine) 2 both undergoes hydrolysis and isomerizes in aqueous acidic solution; however, it is very much more susceptible to hydrolysis than UpU under neutral and mildly basic conditions.
- Liu, Xiaohai,Reese, Colin B.
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p. 3413 - 3416
(2007/10/02)
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- The effect of the 3'-terminal monophosphate group on the metal-ion-promoted hydrolysis of the phosphodiester bonds of short oligonucleotides
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The effect of 3'-terminal monophosphate group on the metal-ion-promoted hydrolysis of the phosphodiester bonds of oligonucleotides has been studied.For this purpose, the rate constants for the hydrolysis of the following oligomers in the presence of Zn2+ and its 1,5,9-triazacyclododecane chelate, Zn2+aneN3, have been determined: (i) ApUpUp(2') and ApUpUp(3'), (ii) Up(Tp)nT (n = 0-4 and 7), (iii) Up(Tp)nTp (n = 0-4 and 7).The results obtained are used to propose a mechanism for the Zn2+ and Zn2+aneN3 promoted hydrolysis of polynucleotides.
- Kuusela, Satu,Azhayev, Alex,Guzaev, Andrei,Loennberg, Harri
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p. 1197 - 1202
(2007/10/02)
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- Oxidation of nucleic acid related compounds by the peroxodisulfate ion
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The treatment of nucleic acid bases, nucleosides, and nucleotides with peroxodisulfate ion in a phosphate buffer solution at pH 7.0 or water at 70-75°C was investigated. The reaction of thymine and 5-methylcytosine nucleosides and nucleotides resulted in the oxidation of the 5-methyl groups. The oxidation products from 1,3-dimethyluracils and the time-course of the reaction of uracils led to two plausible reaction mechanisms for the oxidation of uracils.
- Itahara,Yoshitake,Koga,Nishino
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p. 2257 - 2264
(2007/10/02)
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- Additional Evidence for the Exceptional Mechanism of the Acid-catalysed Hydrolysis of 4-Oxopyrimidine Nucleosides: Hydrolysis of 1-(1-Alkoxyalkyl)uracils, Seconucleosides, 3'-C-Alkyl Nucleosides and Nucleoside 3',5'-Cyclic Monophosphates
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The rate constants for the acid-catalysed hydrolysis of 1-(1-alkoxyethyl)uracils and 1-alkoxymethyluracils have been determined.With both series of compounds, the hydrolysis rate is rather insensitive to the polar nature of the alkoxy group, in striking contrast with the hydrolysis of the corresponding analogues of adenine and cytosine nucleosides, which react via rate-limiting formation of an oxocarbenium ion intermediate.Furthermore, it has been shown that 3',5'-cyclic monophosphates of thymidine and uridine undergo hydrolysis of the N-glycosidic bond 760 and 260 times as fast as their parent nucleosides, while the cyclic monophosphates of 2'-deoxyadenosine and adenosine are depurinated much more slowly than the corresponding nucleosides.On this basis it is suggested that 4-oxopyrimidine nucleosides are hydrolysed by opening of the sugar ring.To obtain further evidence for this exceptional mechanism, comparative kinetic measurements with some seco- and 3'-C-alkyl nucleosides of uracil and adenine have been carried out.
- Oivanen, Mikko,Rajamaeki, Markku,Varila, Jaana,Hovinen, Jari,Mikhailov, Sergey,Loennberg, Harri
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p. 309 - 314
(2007/10/02)
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- Kinetics of Reactions of Pyrimidine Nucleoside 2'- and 3'-Monophosphates under Acidic and Neutral Conditions: Concurrent Phosphate Migration, Dephosphorylation and Deamination
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First-order rate constants for the following reactions of cytidine and uridine monophosphates have been determined over the activity range from pH 7 to H0 -0.7: (i) interconversion of the 2'- and 3'-monophosphates of cytidine and uridine, (ii) dephosphorylation of the 2'-, and 3'- and 5'-monophosphates, and (iii) deamination of cytosine nucleotides to uracil nucleotides.Competition between these reactions under various conditions is discussed, and the data on phosphate migration and phosphoester hydrolysis are compared with those reported earlier for adenosine monophosphates.
- Oivanen, Mikko,Loennberg, Harri
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p. 239 - 242
(2007/10/02)
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- IMMUNOAFFINITY PURIFICATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE FROM LACTUCA COTYLEDONS
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To facilitate further study of a multifunctional phosphodiesterase, previously partially purified from Lactuca cotyledons, a new purification step has been devised.This uses an immunoaffinity column based upon polyclonal antibodies raised against the partially purified enzyme.Preparation of the immunoaffinity column, purufication of the enzyme using the new protocol, and analysis of the activity of the purified enzyme are described.The additional step produced an enzyme preparation with a significantly higher specific activity and free of nucleotidase and non-specific phosphatase activity.The observed properties of the enzyme confirm similarities with mammalian multifunctional phosphodiesterase but reaffirm the existence of two types of substrate binding site on the Lactuca phosphodiesterase.Key Word Index - Lactuca sativa; Compositae; lettuce; cotyledons; cyclic nucleotides; phosphodiesterase; immunoaffinity purification; 3',5'-cyclic AMP; 3',5'-cyclic GMP; 3',5'-cyclic CMP; 3',5'-cyclic UMP.
- Chiatante, Donato,Balconi, Carlotta,Newton, Russell P.,Brown, Eric G.
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p. 2477 - 2484
(2007/10/02)
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- Interaction between 2',5'-A Core Analogues and 2',5'-Specific Phosphodiesterase
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Analogues of (2',5'-)triadenylate (2',5'-A core) were synthesized and their stabilities against 2'-phosphodiesterase (2'PDi) purified from Ehrlich ascites cells were determined.Replacement of the ribose group with an arabinose in different positions of the trimer resulted in dramatic changes in the rate of hydrolysis by 2'PDi.Analogues with an arabinose at the 2' terminal or in he middle position or both were stable against 2'PDi.These stable analogues did not inhibit the degradation of 2',5'-A core by 2'PDi indicating that they did not have the same affinity for the enxyme.Modifications at the 3'-hydroxyls also resulted in structures resistent to degradation by 2'PDi.Replacement of the 6-aminopurine moiety of adenosine by 2-aminopurine resulted in only small changes in stability against 2'PDi.These results show that the configuration of the sugar at 2' and 3' carbons are important for the recognition by 2'-phosphodiesterase.
- Vrang, L.,Drake, A.,Chattopadhyaya, J.,Oeberg, B.
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p. 751 - 760
(2007/10/02)
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