In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3'-5' to 2'-5'

Deoxyribozymes that ligate RNA expand the scope of nucleic acid catalysis and allow preparation of site-specifically modified RNAs. Previously, deoxyribozymes that join a 5'-hydroxyl and a 2',3'-cyclic phosphate were identified by in vitro selection from random DNA pools. Here, the alternative strategy of in vitro evolution was used to transform the 8-17 deoxyribozyme that cleaves RNA into a family of DNA enzymes that ligate RNA. The parent 8-17 DNA enzyme cleaves native 3'-5' phosphodiester linkages but not 2'-5' bonds. Surprisingly, the new deoxyribozymes evolved from 8-17 create only 2'-5' linkages. Thus, reversing the direction of the DNA-mediated process from ligation to cleavage also switches the selectivity in forming the new phosphodiester bond. The same change in selectivity was observed upon evolution of the 10-23 RNA-cleaving deoxyribozyme into an RNA ligase. The DNA enzymes previously isolated from random pools also create 2'-5' linkages. Therefore, deoxyribozyme-mediated formation of a non-native 2'-5' phosphodiester linkage from a 5'-hydroxyl and a 2',3'-cyclic phosphate is strongly favored in many different contexts.     

DIMER PHOTOPRODUCTS IN CYTIDYLYL-(3'-5') CYTIDINE

Abstract— When CpC is exposed to ultraviolet (u.v.) radiation, dimers are produced both photochemically and by dark reactions. A reaction scheme for the production of these dimers is presented. We have shown that a single dimer, CC₁, is initially produced photochemically. This is converted by a spontaneous reaction to a second dimer, CC₂. The dimer CC₂ may also spontaneously revert back to CC₁ and if no side reactions were involved a thermal equilibrium between these txo dimers would be established with about two to three times the concentration of CC₂, as of CC₁. The rate constants for the interconversion of these two dimers vary from about 0.05 to 0.4 hr^-1 over the pH range 2.7 to 8.4. The dimers of CpC have maximum stability near pH 7. The two dimers CC₁ and CC₂ can also be reversed to CpC by u.v. irradiation. Both these dimers in turn deaminate to dimers of UpC and then to UU. The rate constants for these deamination reactions are presented. These lie in the range 0.01 to 0.5 hr^-1 for the pH values studied. Possible structures for the two dimers are suggested.     

Total synthesis of 2',3',4',5',5' '-(2)h(5)-ribonucleosides: the key building blocks for NMR structure elucidation of large RNA

The diastereospecific chemical syntheses of uridine-2',3',4',5',5' '-(2)H(5) (21a), adenosine-2',3',4',5',5' '-(2)H(5) (21b), cytidine-2',3',4',5',5' '-(2)H(5)(2)H(5) (21c), and guanosine-2',3',4',5',5' '-(2)H(5) (21d) (>97 atom % (2)H at C2', C3', C4', and C5'/C5' ') have been achieved for their use in the solution NMR structure determination of oligo-RNA by the Uppsala "NMR-window" concept (refs 4a-c, 5a, 6), in which a small (1)H segment is NMR-visible, while the rest is made NMR-invisible by incorporation of the deuterated blocks 21a-d. The deuterated ribonucleosides 21a-d have been prepared by the condensation of appropriately protected aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-alpha/beta-D-ribofuranose-2,3,4,5,5'-(2)H(5) (19), which has been obtained via diastereospecific deuterium incorporation at the C2 center of appropriate D-ribose-(2)H(4) derivatives either through an oxidation-reduction-inversion sequence or a one-step deuterium-proton exchange in high overall yield (44% and 24%, respectively).     

Synthesis and crystal structure of a salen-type copper(II) complex derived from 3,5'-O-dimethyl-2',3'-diamino-2',3'-dideoxy-beta-D-xylo-uridine

We report the synthesis of the first 2',3'-diamino substituted uridine derivative with xylo-configuration, its use as starting material for the synthesis of salen-type ligands and the first structurally characterised copper(II) complex thereof.     

Linear polymer of a copper(II) complex and its supramolecular selectivity

The supramolecular framework [Cu₂(TU)₄?·?(TAA)₄] _n (TU?=?thiourea; TAA?=?2-(2-imino-4-oxo-5-thiazolidinylidene)acetic anion) has been synthesized by reaction of CuCl₂?·?2H₂O with meso-2,3-dibromosuccinic acid and thiourea. The one-dimensional supramolecular structure of the title complex is constructed through bridge-linkage of the S atoms on the thioureas between adjacent Cu(II) atoms, forming an infinite chain with lots of positive charges. Around the infinite chain, there are four groups of negatively charged hydrogen-bond tubes constructed by four TAA anions. The hydrogen-bond and ionic interactions between adjacent metal-organic polymeric chains and hydrogen-bond tubes form the three-dimensional supramolecular structure of the complex. Supramolecular selectivity from isomers of 2-(2-imino-4-oxo-5-thiazolidinylidene)acetic anion (TAA) has been studied by quantum calculation.     

Trinuclear copper(II) complexes of some aza macrocycles

The syntheses of copper(II) complexes with neutral macrocyclic ligands 1,4,7,10,12,- 15,17,20,23,26,27,30-dodecaazadispiro[10·4·10·4]triacontane (DDST), 2,5,7,10,13,15,18,21,-23,26,29,32-dodecaazatricyclo[20·10·0·0^6,17]dotriacontane (DOCD) and 2,5,7,10,13,16,18,-21,23,26,29,32-dodecaaza-1,6,17,22-tetrachlorotricyclo[20·10·0·0^6,17]dotriacontane (DTTD) derived from triethylenetetramine, 1,2-diaminoethane and chlorocarbons (carbon tetrachloride, 1,l,2,2-tetrachloroethane and hexachloroethane, respectively) have been studied. Complexes [Cu₃(DDST)]Cl₆, [Cu₃(DOCD)]Cl₆ and [Cu₃(DTTD)]Cl₆?·?H₂O and the copper ion-free ligand hydrochlorides DDST?·?12HCl and DOCD?·?12HCl are supported by elemental analyses, conductivity measurements and spectroscopic studies. Potentiometric equilibrium studies on DDST and DOCD hydrochlorides and their copper complexes also support the structures.     

3-(2-二唑啉基)-色酮类化合物的合成研究

报道了取代的邻羟基苯乙酮(1a～1e)经Vilsmeier-Haack反应一步制得3-醛基色酮(2a～2e),2a～2e与取代的芳酰肼(3a～3c)缩合制得15个酰腙类化合物(4a～4o),4a～4o在醋酸酐作用下关环得15个3-(2-苝二唑啉基)-色酮(5a～5o),并通过元素分析,IR,~1H NMR和MS诺数据确证了上述化合物的结构。     

The photochemistry of thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine in aqueous solution

The photochemistry of the dinucleoside monophosphate thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine (Tpm5dC) has been studied in aqueous solution using both 254 nm and UV-B radiation. A variety of dinucleotide photoproducts containing 5-methylcytosine (m5C) have been isolated and characterized. These include two cyclobutane dimers (CBD) (the cis-syn [c,s]and trans-syn forms), a (6-4) adduct and its related Dewar isomer, and two isomers of a product in which the m5C moiety was converted into an acrylamidine. Small amounts of thymidylyl-(3'-5')-thymidine (TpT) were also formed, presumably as a secondary photoreaction product. In addition, a photoproduct was characterized in which the m5C moiety was lost, thus generating 3'-thymidylic acid esterified with 2'-deoxyribose at the 5-hydroxyl on the sugar moiety. The c,s CBD of Tpm5dC readily undergoes deamination to form the corresponding CBD of TpT. The kinetics of this deamination process has been studied; the corresponding enthalpy and entropy of activation for the reaction have been evaluated at pH 7.4 as being, respectively, 73.4 kJ/mol and -103.5 J/K mol. Deamination was not observed for the other characterized photoproducts of Tpm5dC.     

Adsorption voltammetry of the copper(II) 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complex

The voltammetric determination of copper(II), based on adsorptive accumulation of the Cu(II)-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (S-Br-PADAP) complex on a hanging mercury drop electrode, is reported. The complex can be accumulated at the electrode at constant potential in 0.1M ammonium nitrate/ammonia buffer solution, and its reduction wave observed by scanning the potential in the negative direction, in the differential pulse mode. The calibration graph for copper is linear over the range 0.05-0.5muM, with accumulation for 5 min at -0.20 V. The adsorption of the complex is discussed and compared with that of copper complexes with several other pyridylazo derivatives.     

Trinuclear copper(I) complex of 1,3-bis(2-pyridinylmethyl)imidazolylidene as a carbene-transfer reagent for the preparation of catalytically active nickel(II) and palladium(II) complexes

Reactions of 1,3-bis(pyridin-2-ylmethyl)-1H-imidazol-3-ium hexafluorophosphate, ([HL1](PF₆), L1 = 1,3-bis(pyridin-2-ylmethyl)imidazolylidene) and 1,3-bis(pyridin-2-ylmethyl)-1H-benzimidazol-3-ium hexafluorophosphate ([HL2](PF₆), L2 = 1,3-bis(pyridin-2-ylmethyl)benzoimidazolylidene) with cuprous oxide in acetonitrile readily yielded trinuclear complexes [Cu₃(L1)₃(PF₆)₃] (1) and [Cu₃(L2)₃(PF₆)₃] (2). Treatment of 1 with Ni(PPh₃)₂Cl₂ and Pd(cod)Cl₂ gave [Ni(L1)Cl](PF₆) (3) and [Pd(L1)Cl](PF₆) (4), respectively, due to transmetalation. [Ni(L1)₂](PF₆)₂ (5) was obtained from the reaction of [Cu₃(L1)₃(PF₆)₃] and Raney nickel in acetonitrile. All these complexes have been fully characterized. Both 1 and 2 consist of a triangular Cu₃ core with each Cu–Cu bond capped by an imidazolylidene group. Each imidazolylidene acts as a bridging ligand in a μ₂ mode and is bonded equally to two Cu(I) ions. The pincer nickel and palladium complexes are square-planar and contain a tridentate NCN ligand. Complexes 3 and 4 are efficient catalyst precursors for Kumada–Corriu and Suzuki–Miyaura coupling reactions of aryl halides with organometallic reagents.     

Crystal structure of the complex of copper(II) with 3-(2-hydroxy-3-sulfo-5-nitrophenylazo)-pentadione-2,4

A new complex of copper (II) with 3-(2-hydroxy-3-sulfo-5-nitrophenylazo)-pentadione-2,4 (HL) is synthesized and analyzed by single crystal XRD. The crystals are monoclinic: a = 7.7563(4) Å, b = 24.0157(12) Å, c = 24.6353(13) Å, β = 91.9490(10)°, V = 4586.2(4) ų, space group P2₁/c, Z = 2, ρ_calc = 1.713 g/cm³, R = 0.0695. The environment of the copper atom is formed by three oxygen atoms, the nitrogen atom of tetradentate ligand, and the oxygen atom of a water molecule.     

Trinuclear copper(I) complex of 1,3-bis(2-pyridinylmethyl)imidazolylidene as a carbene-transfer reagent for the preparation of catalytically active nickel(II) and palladium(II) complexes

Reactions of 1,3-bis(pyridin-2-ylmethyl)-1H-imidazol-3-ium hexafluorophosphate, ([HL1](PF₆), L1 = 1,3-bis(pyridin-2-ylmethyl)imidazolylidene) and 1,3-bis(pyridin-2-ylmethyl)-1H-benzimidazol-3-ium hexafluorophosphate ([HL2](PF₆), L2 = 1,3-bis(pyridin-2-ylmethyl)benzoimidazolylidene) with cuprous oxide in acetonitrile readily yielded trinuclear complexes [Cu₃(L1)₃(PF₆)₃] (1) and [Cu₃(L2)₃(PF₆)₃] (2). Treatment of 1 with Ni(PPh₃)₂Cl₂ and Pd(cod)Cl₂ gave [Ni(L1)Cl](PF₆) (3) and [Pd(L1)Cl](PF₆) (4), respectively, due to transmetalation. [Ni(L1)₂](PF₆)₂ (5) was obtained from the reaction of [Cu₃(L1)₃(PF₆)₃] and Raney nickel in acetonitrile. All these complexes have been fully characterized. Both 1 and 2 consist of a triangular Cu₃ core with each Cu–Cu bond capped by an imidazolylidene group. Each imidazolylidene acts as a bridging ligand in a μ₂ mode and is bonded equally to two Cu(I) ions. The pincer nickel and palladium complexes are square-planar and contain a tridentate NCN ligand. Complexes 3 and 4 are efficient catalyst precursors for Kumada–Corriu and Suzuki–Miyaura coupling reactions of aryl halides with organometallic reagents.     

Sugar conformational effects on the photochemistry of thymidylyl(3'-5')thymidine

The synthesis and conformational analysis of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a), the analogue of thymidylyl(3'-5')thymidine (TpT; 1b) in which a methoxy group replaces each 2'-alpha-hydrogen atom, are described. In comparison with TpT, such modification increases the population of the C3'-endo conformer of the sugar ring puckering at the 5'- and 3'-ends from 30 to 75% and from 37 to 66%, respectively. Photolyses of 1a and TpT at 254 nm are qualitatively comparable (the cis-syn cyclobutane pyrimidine dimer and the (6-4) photoproduct are formed), although it is significantly faster in the case of 1a. These results are explained by the increased propensity of the modified dinucleotide to adopt a base-stacked conformation geometry reminiscent of that for TpT.     

Highly efficient phosphodiester hydrolysis promoted by a dinuclear copper(II) complex

The interaction of Cu(II) with the ligand tdci (1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol) was studied both in the solid state and in solution. The complexes that were formed were also tested for phosphoesterase activity. The pentanuclear complex [Cu(5)(tdciH(-2))(tdci)(2)(OH)(2)(NO(3))(2)](NO(3))(4).6H(2)O consists of two dinuclear units and one trinuclear unit, having two shared copper(II) ions. The metal centers within the pentanuclear structure have three distinct coordination environments. All five copper(II) ions are linked by hydroxo/alkoxo bridges forming a Cu(5)O(6) cage. The Cu-Cu separations of the bridged centers are between 2.916 and 3.782 A, while those of the nonbridged metal ions are 5.455-5.712 A. The solution equilibria in the Cu(II)-tdci system proved to be extremely complicated. Depending on the pH and metal-to-ligand ratio, several differently deprotonated mono-, di-, and trinuclear complexes are formed. Their presence in solution was supported by mass, CW, and pulse EPR spectroscopic study, too. In these complexes, the metal ions are presumed to occupy tridentate [O(ax),N(eq),O(ax)] coordination sites and the O-donors of tdci may serve as bridging units between two metal ions. Additionally, deprotonation of the metal-bound water molecules may occur. The dinuclear Cu(2)LH(-3) species, formed around pH 8.5, provides outstanding rate acceleration for the hydrolysis of the activated phosphodiester bis(4-nitrophenyl)phosphate (BNPP). The second-order rate constant of BNPP hydrolysis promoted by the dinuclear complex (T = 298 K) is 0.95 M(-1) s(-1), which is ca. 47600-fold higher than that of the hydroxide ion catalyzed hydrolysis (k(OH)). Its activity is selective for the phosphodiester, and the hydrolysis was proved to be catalytic. The proposed bifunctional mechanism of the hydrolysis includes double Lewis acid activation and intramolecular nucleophilic catalysis.