Trinuclear copper(II) complex showing high selectivity for the hydrolysis of 2'-5' over 3'-5' for UpU and 3'-5' over 2'-5' for ApA ribonucleotides

The cooperative action of multiple Cu(II) nuclear centers is shown to be effective and selective in the hydrolysis of 2'-5' and 3'-5' ribonucleotides. Reported herein is the specific catalysis by two trinuclear Cu(II) complexes of L3A and L3B. Pseudo first-order kinetic studies reveal that the L3A trinuclear Cu(II) complex effects hydrolysis of Up(2'-5')U with a rate constant of 28 x 10(-)(4) min(-)(1) and Up(3'-5')U with a rate constant of 0.5 x 10(-)(4) min(-)(1). The hydrolyses of Ap(3'-5')A and Ap(2'-5')A proceed with rate constants of 24 x 10(-)(4) min(-)(1) and 0.5 x 10(-)(4) min(-)(1) respectively. The L3A trinuclear Cu(II) complex demonstrates high specificity for Up(2'-5')U and Ap(3'-5')A. Similar studies with the more rigid L3B trinuclear Cu(II) complex shows no selectivity and yields lower rate constants for hydrolysis. The selectivity observed with the L3A ligand is attributed to the geometry of the ligand-bound diribonucleotide which ultimately dictates the proximity of the attacking hydroxyl and the phosphoester to a Cu(II) center for activation and subsequent hydrolysis.     

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.     

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).     

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.     

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.     

Deoxyribozymes with 2'-5' RNA ligase activity

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.     

Nonenzymatic, template-directed ligation of oligoribonucleotides is highly regioselective for the formation of 3'-5' phosphodiester bonds

We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'-5' rather than 2'-5' phosphodiester bonds. Formation of the 3'-5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'-5'- to 2'-5'-linked products was 10-15:1 when the 5'-phosphate was activated as the imidazolide, and 60-80:1 when the 5'-phosphate was activated by the formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'-5'-linkages in oligonucleotide ligation is primarily due to occurence of ligation within the context of an extended Watston-Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.     

Synthesis and mesomorphic properties of 4-methoxyphenyl 4'- n -alkoxy-2',3',5',6'-tetrafluorobiphenyl-1-carboxylates

A novel type of 4-methoxyphenyl 4'- n -alkoxy-2',3',5',6'-tetrafluorobiphenyl-1-carboxylates have been synthesized. Textural observations by polarizing microscopy and DSC measurements of the phase transitions show that all of these compounds are thermotropic liquid crystals with only a nematic mesophase. The relationship between the properties and chemical structures of these compounds is discussed.     

酰氨基硫脲及相关杂环衍生物的研究XXIX: 2-(3'-溴苯胺基)-5-[5'-氨基-1'-(4"-氯苯基)-1',2',3'-三唑-4'-基]-1,3,4-噻二唑的晶体结构

经1-[5'-氨基-1'-(4"-氯苯基)-1',2',3'-三唑-4'-甲酰基]-4-(3'-溴苯基)-3-氨基硫脲在浓硫酸作用下制得2-(3'-溴苯胺基)-5-[5'-氨基-1'-(4"-氯苯基)-1',2',3'-三唑-4'-基]-1,3,4-噻二唑化合物。该化合物的晶体结构经X射线衍射分析确定, 化合物属三斜晶系, P1空间群, a=1.1784(2), b=1.4455(2),c=1.1353(1)nm; α=100.68(1), β=109.50(1), γ=79.89(1)°; V=1.7779nm^3; 分子式C~1~6H~1~1BrClN~7S, Mr=448.75; Dc=1.673g/cm^3, Z=4,μ=58.16cm^-^1, 最终偏离因子R=0.084, Rw=0.086。分析化合物的键长, 键角数据表明, 该分子具有离域π键结构。     

General deoxyribozyme-catalyzed synthesis of native 3'-5' RNA linkages

An elusive goal for nucleic acid enzymology has been deoxyribozymes that ligate RNA rapidly, sequence-generally, with formation of native 3'-5' linkages, and in preparatively useful yield. Using in vitro selection, we have identified Mg2+- and Zn2+-dependent deoxyribozymes that simultaneously fulfill all four of these criteria. The new deoxyribozymes operate under practical incubation conditions and have modest RNA substrate sequence requirements, specifically D downward arrowRA for 9DB1 and A downward arrowR for 7DE5 (D = A, G, or U; R = A or G). These requirements are comparable to those of deoxyribozymes such as 10-23 and 8-17, which are already widely used as biochemical tools for RNA cleavage. We anticipate that the 9DB1 and 7DE5 deoxyribozymes will find immediate practical application for RNA ligation.     

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.     

Photoreaction at 5'-(G/C)AA(Br)UT-3' sequence in duplex DNA: efficient generation of uracil-5-yl radical by charge transfer

The photoreactivities of 5-halouracil-containing DNA have widely been used for analysis of protein-DNA interactions and have recently been used for probing charge-transfer processes along DNA. Despite such practical usefulness, the detailed mechanisms of the photochemistry of 5-halouracil-containing DNA are not well understood. We recently discovered that photoirradiation of BrU-substituted DNA efficiently produced 2'-deoxyribonolactone at 5'-(G/C)AABrUBrU-3' and 5'-(G/C)ABrUBrU-3' sequences in duplex DNA. Using synthetic oligonucleotides, we found that similar photoreactivities were maintained at the 5'-(G/C)AABrUT-3' sequence, providing ribonolactone as a major product with concomitant release of adenine base. In this paper, the photoreactivities of various oligonucleotides possessing the 5'-BrUT-3' sequence were examined to elucidate the essential factors of this photoreaction. HPLC product analysis indicated that the yield of 2'-deoxyribonolactone largely depends on the ionization potential of the purine derivatives located 5'-upstream of 5'-BrUT-3', as well as the electron-donating ability of their pairing cytosine derivatives. Oligonucleotides that possess G in the complementary strand provided the ribonolactone with almost the same efficiency. These results clearly suggest that the photoinduced charge transfer from the G-5' upstream of 5'-BrUT-3' sequence, in the same strand and the complementary strand, initiates the reaction. To examine the role of intervening A/T base pair(s) between the G/C and the 5'-BrUT-3' sequence, the photoreactivities of a series of oligonucleotides with different numbers of intervening A/T base pairs were examined. The results revealed that the hotspot sequence consists of the electron-donating G/C base pair, the 5'-BrUT-3' sequence as an acceptor, and an appropriate number of A/T base pairs as a bridge for the charge-transfer process.     

Investigations on the interferon-induced 2'-5' oligoadenylate system using analytical capillary isotachophoresis

The activity of the interferon inducible enzyme 2'-5' oligoadenylate synthetase (2-5A synthetase, E.C. 2.7.7) which converts ATP into a series of 2'-5' oligoadenylates was measured using analytical capillary isotachophoresis. The turnover rate of ATP during the reaction was monitored by determination of its concentration at the beginning and the end of the 2-5A synthetase reaction. The enzyme was analysed in extracts of peripheral blood mononuclear cells either pretreated or not with interferon.     

Matrix-assisted laser desorption/ionization detection of polymerase chain reaction products by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase

The 5'-3' exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5'-3' exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo-taq assay or probe degradation assay. This method should be amenable to automation.     

Crystal structure and photochemical behavior in solution of the 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine

The 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine (TnsoT, 1) exhibits a preference for a C3'-endo conformation in the solution and solid states. Its photochemical behavior in solution is compared to that of its natural counterpart, thymidylyl(3'-5')thymidine (TpT, 2), to get further insight into the significance of the C3'-endo conformation on the photoproduct formation at the single-stranded dinucleotide level. Irradiation at 254 nm of 1 led to the same type of photoproducts as observed with 2. However, 1 was significantly more photoreactive than 2, and accordingly, the initial rate of photoproduct formation was enhanced in accordance with its propensity to base stack compared to 2. The corresponding quantum yields were determined and showed that the enhancement factor (1 compared to 2) is moderate for the cyclobutane pyrimidine dimer (CPD) (1.26) and much higher for the (6-4) photoproduct (1.8). These data strongly suggest that the CPD and (6-4) photoproduct arise from distinct minor stacked conformations.     

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine)

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine) have been followed by RP HPLC over a wide pH range at 363.2 K in order to elucidate the role of the 2'-hydroxyl group as a hydrogen-bond donor upon departure of the 3'-uridine moiety. Under neutral and basic conditions, guanosyl-(3',3')-uridine undergoes hydroxide ion-catalyzed cleavage (first order in [OH(-)]) of the P-O3' bonds, giving uridine and guanosine 2',3'-cyclic monophosphates, which are subsequently hydrolyzed to a mixture of 2'- and 3'-monophosphates. This bond rupture is 23 times as fast as the corresponding cleavage of the P-O3' bond of guanosyl-(3',3')-(2',5'-di-O-methyluridine) to yield 2',5'-O-dimethyluridine and guanosine 2',3'-cyclic phosphate. Under acidic conditions, where the reactivity differences are smaller, depurination and isomerization compete with the cleavage. The effect of Zn(2+) on the cleavage of the P-O3' bonds of guanosyl-(3',3')-uridine is modest: about 6-fold acceleration was observed at [Zn(2+)] = 5 mmol L(-)(1) and pH 5.6. With guanosyl-(3',3')-(2',5'-di-O-methyluridine) the rate-acceleration effect is greater: a 37-fold acceleration was observed. The mechanisms of the partial reactions, in particular the effects of the 2'-hydroxyl group on the departure of the 3'-linked nucleoside, are discussed.     

Synthesis of some 3',5'-dideoxy-5'-C-phosphonomethyl nucleosides.

Ammonium [1-(3',5',6'-trideoxy-beta-D-erythro-hexofuranosyl)thymine]-6'- phosphonate (1), ammonium 3',5'-dideoxycytidine-5'-C-methylphosphonate (2) and 3',5'-dideoxyadenosine-5'-C-methyl phosphonic acid (3) have been synthesized and tested for anti-HIV activity. The key steps involved an Arbuzov reaction between triethyl phosphite and 3,5,6-trideoxy-6-iodo-1,2-O-isopropylidene-alpha-D-erythro- hexofuranose (7), followed by condensation with the appropriate nucleoside bases. The substances 1, 2 and 3 have been tested in vitro against HIV.     

Synthesis of 3,4,4'- and 2',3,4-Triaminodiphenyl Ethers

Procedures were developed for preparing 3,4,4'- and 2',3,4-triaminodiphenyl ethers by the reaction of 4-acetylaminophenol with, respectively, 4-nitro- or 2-nitrochlorobenzene, followed by nitration of the resulting 4-acetylamino-4'- or -2'-nitrodiphenyl ether, hydrolysis of the product, 4-acetylamino-3,4'- or -2',3-dinitrodiphenyl ether, and reduction of 4-amino-3,4'- or -2'-3-dinitrodiphenyl ether.     

Thio effects on the departure of the 3'-linked ribonucleoside from diribonucleoside 3',3'-phosphorodithioate diesters and triribonucleoside 3',3',5'-phosphoromonothioate triesters: implications for ribozyme catalysis

To provide a solid chemical basis for the mechanistic interpretations of the thio effects observed for large ribozymes, the cleavage of triribonucleoside 3',3',5'-phosphoromonothioate triesters and diribonucleoside 3',3'-phosphorodithioate diesters has been studied. To elucidate the role of the neighboring hydroxy group of the departing 3'-linked nucleoside, hydrolysis of 2',3'-O-methyleneadenosin-5'-yl bis[5'-O-methyluridin-3'-yl] phosphoromonothioate (1 a) has been compared to the hydrolysis of 2',3'-O-methyleneadenosin-5'-yl 5'-O-methyluridin-3'-yl 2',5'-di-O-methyluridin-3'-yl phosphoromonothioate (1 b) and the hydrolysis of bis[uridin-3'-yl] phosphorodithioate (2 a) to the hydrolysis of uridin-3'-yl 2',5'-di-O-methyluridin-3'-yl phosphorodithioate (2 b). The reactions have been followed by RP HPLC over a wide pH range. The phosphoromonothioate triesters 1 a,b undergo two competing reactions: the starting material is cleaved to a mixture of 3',3'- and 3',5'-diesters, and isomerized to 2',3',5'- and 2',2',5'-triesters. With phosphorodithioate diesters 2 a,b, hydroxide-ion-catalyzed cleavage of the P--O3' bond is the only reaction detected at pH >6, but under more acidic conditions desulfurization starts to compete with the cleavage. The 3',3'-diesters do not undergo isomerization. The hydroxide-ion-catalyzed cleavage reaction with both 1 a and 2 a is 27 times as fast as that compared with their 2'-O-methylated counterparts 1 b and 2 b. The hydroxide-ion-catalyzed isomerization of the 3',3',5'-triester to 2',3',5'- and 2',2',5'-triesters with 1 a is 11 times as fast as that compared with 1 b. These accelerations have been accounted for by stabilization of the anionic phosphorane intermediate by hydrogen bonding with the 2'-hydroxy function. Thio substitution of the nonbridging oxygens has an almost negligible influence on the cleavage of 3',3'-diesters 2 a,b, but the hydrolysis of phosphoromonothioate triesters 1 a,b exhibits a sizable thio effect, k(PO)/k(PS)=19. The effects of metal ions on the rate of the cleavage of diesters and triesters have been studied and discussed in terms of the suggested hydrogen-bond stabilization of the thiophosphorane intermediates derived from 1 a and 2 a.     

Efficient generation of 2'-deoxyuridin-5-yl at 5'-(G/C)AA(X)U(X)U-3' (X = Br, I) sequences in duplex DNA under UV irradiation

The photoreactivity of 5-halouracil-containing DNA was investigated using 450 base pair DNA fragments under 302 nm irradiation. Heat-dependent cleavage selectively occurs at 5'-(G/C)AAXUXU-3' and 5'-(G/C)AXUXU-3' (X = Br, I) sequences in double-stranded DNA. HPLC product analysis indicated that 2'-deoxyribonolactone residues are effectively generated at these sequences. These observations will be useful in studying the molecular basis of the sequence-dependent DNA-damaging process in UV-irradiated 5-halouracil-containing DNA.