Hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine: kinetics and mechanisms for the cleavage, desulfurization, and isomerization of the internucleosidic linkage

The hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine [3',5'-Up(s)2U] were followed by HPLC over a wide pH range at 363.2 K. Under acidic and neutral conditions, three reactions compete: (i) desulfurization to a mixture of the (Rp)- and (Sp)-diastereomers of the corresponding 3',5'- and 2',5'-phosphoromonothioates [3',5'- and 2',5'-Up(s)U], which are subsequently desulfurized to a mixture of uridylyl(3',5')- and -(2',5')uridine [3',5'- and 2',5'-UpU], (ii) isomerization to 2',5'-Up(s)2U, and (iii) cleavage to uridine, in all likelihood via a 2',3'-cyclic phosphorodithioate (2',3'-cUMPS2). Under alkaline conditions (pH > 8), only a hydroxide ion catalyzed hydrolysis to uridine via 2',3'-cUMPS2 takes place. At pH 3-7, all three reactions are pH-independent, the desulfurization being approximately 1 order of magnitude faster than the cleavage and isomerization. At pH < 3, all the reactions are hydronium ion catalyzed. On going to very acidic solutions, the cleavage gradually takes over the desulfurization and isomerization. Accordingly, the cleavage overwhelmingly predominates at pH < 0. The overall hydrolytic stability of 3',5'-Up(s)2U is comparable to that of (Sp)- and (Rp)-3',5'-Up(s)U (and to that of 3',5'-UpU, except at pH < 2). The rate of the hydroxide ion catalyzed hydrolysis of 3',5'-Up(s)2U is 37% and 53% of that of (Sp)- and (Rp)-3',5'-Up(s)U, respectively. The reactions, however, differ with the respect of the product accumulation. While the phosphoromonothioates produce a mixture of 2'- and 3'-thiophosphates as stable products, 3',5'-Up(s)2U is hydrolyzed to uridine without accumulation of the corresponding dithiophosphates. At pH < 3, where the hydrolysis is hydronium ion catalyzed, the kinetic thio-effect of the second thio substitution is small: under very acidic conditions (Ho -0.69), (Sp)-3',5'-Up(s)U reacts 1.6 times as fast as 3',5'-Up(s)2U, but the reactivity difference decreases on going to less acidic solutions. In summary, the hydrolytic stability of 3',5'-Up(s)2U closely resembles that of the corresponding phosphoromonothioate. While replacing one of the nonbridging phosphate oxygens of 3',5'-UpU with sulfur stabilizes the phosphodiester bond under acidic conditions by more than 1 order of magnitude, the replacement of the remaining nonbridging oxygen has only a minor influence on the overall hydrolytic stability.     

5′-Phosphouridine as a source of terminal phosphate groups in oligonucleotides

A universal key component is proposed for the preparation of oligonucleotides with 3′- and 5′-terminal phosphate groups — 2′,3′-dibenzoyluridin-5′-yl (4-chlorophenylphosphate) (pU(Bz)₂), which is a potential source of the phosphate group. The condensation ofpU(Bz)₂ with the 5′-OH or the 3′-OH group of a protected oligonucleotide leads to the formation of oligodeoxyribonucleotides with 5′- or 3′-terminal uridine, respectively. The oxidation of the 2′,3′-cis-glycol group of the terminal uridine unit followed by β-elimination forms oligodeoxyribonucleotides with terminal phosphate groups.     

Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexyl phosphates as substituents

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5' termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5'-hydroxyl group maximized their short RNA selectivity. The 2'-O-methyl-RNA (5'-XC(m)A(m)A(m)C(m)C(m)U(m)A(m)C(m)U(m)) having these modifications exhibits ca. 10 °C higher T(m) in the duplexes with the complementary short RNA (3'-GUUGGAUGA-5') than with the long RNA (3'-AUUAUAUGUUGGAUGAUGGUUA-5'). The oligodeoxynucleotides having the same modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.     

MONOCLONAL ANTIBODY WITH SPECIFIC AFFINITY FOR PYRIMIDINE DIMERS IN RNA

Abstract— A monoclonal antibody was prepared which meets three criteria for specific binding to pyrimidine dimers in RNA. (i) UV irradiation at wavelengths greater than 300 nm in the presence of a triplet state sensitizer, or at 270 nm without sensitizer, promotes antibody binding to RNA and polyribonucleotides, (ii) Antibody binding is reduced by exposure to UV radiation of short wavelength (240 nm) following sensitized irradiation (<300 nm). (iii) Antibody binding is dependent upon the presence of adjacent pyrimidine ribonucleotides. The antibody recognizes a single uridine dimer with one or more additional nucleotides at both ends. Affinity for a single uridine dimer with additional nucleotides at only the 3' end is substantially weaker.     

A dizinc complex for selective fluorescence sensing of uridine and uridine-containing dinucleotides

A dizinc complex with a polyamine macrocycle is able to selectively bind and sense uridine (U) as well as the uridine-containing ribodinucleotides U(3'-5')pU and U(3'-5')pA, thanks to an exciplex emission arising from a pi-stacked complex involving the dipyridine unit and Zn(II)-bound uridine moieties.     

Duplex and triplex formation of mixed pyrimidine oligonucleotides with stacking of phenyl-triazole moieties in the major groove

5-(1-Phenyl-1,2,3-triazol-4-yl)-2'-deoxycytidine was synthesized from a modified CuAAC protocol and incorporated into mixed pyrimidine oligonucleotide sequences together with the corresponding 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine. With consecutive incorporations of the two modified nucleosides, improved duplex formation with a complementary RNA and improved triplex formation with a complementary DNA duplex were observed. The improvement is due to π-π stacking of the phenyl-triazole moieties in the major groove. The strongest stacking and most pronounced positive influence on thermal stability was found in between the uridine analogues or with the cytidine analogue placed in the 3' direction to the uridine analogue. Modeling indicated a different orientation of the phenyl-triazole moieties in the major groove to account for the difference between the two nucleotides. The modified oligonucleotides were all found to be significantly stabilized toward nucleolytic degration.     

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.     

Sugar radicals formed by photoexcitation of guanine cation radical in oligonucleotides

This work presents evidence that photoexcitation of guanine cation radical (G+*) in dGpdG and DNA-oligonucleotides TGT, TGGT, TGGGT, TTGTT, TTGGTT, TTGGTTGGTT, AGA, and AGGGA in frozen glassy aqueous solutions at low temperatures leads to hole transfer to the sugar phosphate backbone and results in high yields of deoxyribose radicals. In this series of oligonucleotides, we find that G+* on photoexcitation at 143 K leads to the formation of predominantly C5'* and C1'* with small amounts of C3'*. Photoconversion yields of G+* to sugar radicals in oligonucleotides decreased as the overall chain length increased. However, for high molecular weight dsDNA (salmon testes) in frozen aqueous solutions, substantial conversion of G+* to C1'* (only) sugar radical is still found (ca. 50%). Within the cohort of sugar radicals formed, we find a relative increase in the formation of C1'* with length of the oligonucleotide, along with decreases in C3'* and C5'*. For dsDNA in frozen solutions, only the formation of C1'* is found via photoexcitation of G+*, without a significant temperature dependence (77-180 K). Long wavelength visible light (>540 nm) is observed to be about as effective as light under 540 nm for photoconversion of G+* to sugar radicals for short oligonucleotides but gradually loses effectiveness with chain length. This wavelength dependence is attributed to base-to-base hole transfer for wavelengths >540 nm. Base-to-sugar hole transfer is suggested to dominate under 540 nm. These results may have implications for a number of investigations of hole transfer through DNA in which DNA holes are subjected to continuous visible illumination.     

THE PHOTOPHYSICAL AND PHOTOCHEMICAL PROCESSES OF TRYPTOPHAN IN INTERACTION WITH POLYNUCLEOTIDES: LASER FLASH PHOTOLYSIS STUDY

Abstract— The influence of nucleotides or polynucleotides on the photophysics and the photochemistry of tryptophan (Trp) derivatives has been investigated in aqueous solutions using the 265 nm laser flash photolysis technique. In solutions containing mixtures of N -acetyltryptophanamide and uridine monophosphate (UMP) or mercurated dUMP, the Trp triplet and the hydrated electron (e_aq) are quenched at almost diffusion controlled rates by the nucleotides leading to uracil reduction. Lysyl-tryptophyl-α-lysine (Lys-Trp-Lys) forms stable complexes in solution with normal or mercurated poly(uridylic acid) [poly(U)]. In the Poly(rU)-Lys-Trp-Lys complex the Trp triplet state is completely quenched, whereas the Trp triplet formation quantum yield is enhanced in complexes with mercurated poly(U). In this last case, the 'heavy atom effect' is characterized by a shortening of the Trp triplet lifetime in agreement with low temperature experiments. Our results also show that photoionization of Trp does occur in the complexed state with both polymers. The e_aq lifetime is however longer with the complexed than with the free peptide.     

应用^3^1P NMR研究RNase A水解核糖核酸的机制

核糖核酸酶A(RNase A)水解核糖核酸的机制前人已有研究.Markham等和Brown等根据水解过程中有2′,3′-环核苷酸的生成提出了两步机制,即磷酰基转移和水解开环.Witzel等用紫外差值(ΔA_(286))光谱和pH-stat(pH恒定器)技术进行动力学研究,所得的结果支持了上述机制.Williams用同样的方法进行研究,提出除了两步机制外,还有另一途径,即二核苷(3′→5′)单磷酸二酯(A)不经过2′,3′-环核苷酸(B)而直接转化为3′-核苷酸     

Effects of the interaction between beta-carboline-3-carboxylic acid N-methylamide and polynucleotides on singlet oxygen quantum yield and DNA oxidative damage

The complexation of beta-carboline-3-carboxylic acid N-methylamide (betaCMAM) with the sodium salts of the nucleotides polyadenylic (Poly A), polycytidylic (Poly C), polyguanylic (Poly G), polythymidylic (Poly T) and polyuridylic (Poly U) acids, and with double stranded (dsDNA) and single stranded deoxyribonucleic acids (ssDNA) was studied at pH 4, 6 and 9. Predominant 1:1 complex formation is indicated from Job plots. Association constants were determined using the Benesi-Hildebrand equation. BetaCMAM-sensitized singlet oxygen quantum yields were determined at pH 4, 6 and 9, and the effects on this of adding oligonucleotides, dsDNA and ssDNA were studied at the three pH values. With dsDNA, the effect on betaCMAM triplet state formation was also determined through triplet-triplet transient absorption spectra. To evaluate possible oxidative damage of DNA following singlet oxygen betaCMAM photosensitization, we used thiobarbituric acid-reactivity assays and electrophoretic separation of DNA assays. The results showed no oxidative damage at the level of DNA degradation or strand break.     

Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines.

A heptamer composed of C5-(1-propynyl) pyrimidines (Y(p)'s) is a potent and specific antisense agent against the mRNA of SV40 large T antigen (Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.; Froehler, B. C. Nat. Biotechnol. 1996, 14, 840-844). To characterize the role of the propynyl groups in molecular recognition, thermodynamic increments associated with substitutions in DNA:RNA duplexes, such as 5'-dCCUCCUU-3':3'-rGAGGAGGAAAU-5', have been measured by UV melting experiments. For nucleotides tested, an unpaired dangling end stabilizes unmodified and propynylated duplexes similarly, except that addition of a 5' unpaired rA is 1.4 kcal/mol more stabilizing on the propynylated, PODN:RNA, duplex than on the DNA:RNA duplex. Free energy increments for addition of single propynyl groups range from 0 to -4.0 kcal/mol, depending on the final number and locations of substitutions. A preliminary model for predicting the stabilities of Y(p)-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5'-dC(p)C(p)U(p)C(p)C(p)U(p)U(p)-3':3'-rGAGIAGGAAAU-5', destabilizes the duplex by 3.9 kcal/mol, compared to 1.7 kcal/mol for the same change within the unpropynylated duplex. This 2.2 kcal/mol difference is eliminated by removing a single propynyl group three base pairs away. CD spectra suggest that single propynyl deletions within the PODN:RNA duplex have position-dependent effects on helix geometry. The results suggest long-range cooperativity between propynyl groups and provide insights for rationally programming oligonucleotides with enhanced binding and specificity. This can be exploited in developing technologies that are dependent upon nucleic acid-based molecular recognition.     

Synthesis of oligonucleotides with 3'-terminal 5-(3-acylamidopropargyl)-3'-amino-2',3'-dideoxyuridine residues and their reactivity in single-nucleotide steps of chemical replication

Oligonucleotides with a 3'-terminal 5-alkynyl-3'-amino-2',3'-dideoxyuridine residue were prepared, starting from 2'-deoxyuridine. The optimized route employs a 2',3'-dideoxy-3'-trifluoroacetamido-5-iodouridine 5'-phosphoramidite as building block for DNA synthesis and involves on-support Sonogashira coupling with N-tritylpropargylamine to generate oligonucleotides. The amino group of the propargylamine side chain was acylated to accelerate primer extension reactions involving the 3'-amino group. Three acyl groups were identified that decrease the half-life for DNA-templated extension steps with 7-azabenzotriazole esters of 2'-deoxynucleotides. The residue of 4-pyrenylbutyric acid was found to accelerate primer extension reactions and to render them more selective than those of the control primer. With this substituent, primer extension is also faster than previously measured for three-strand systems involving template, aminoprimer, and a downstream-binding helper oligonucleotide. Fast-reacting primers might become useful for genotyping single nucleotides.     

The pseudouridine synthases: revisiting a mechanism that seemed settled

RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.     

Convertible and Constrained Nucleotides: The 2’-Deoxyribose 5’-C-Functionalization Approach,a French Touch

Many strategies have been developed to modulate the biological or biotechnical properties of oligonucleotides by introducing new chemical functionalities or by enhancing their affinity and specificity while restricting their conformational space. Among them, we review our approach consisting of modifications of the 5’-C-position of the nucleoside sugar. This allows the introduction of an additional chemical handle at any position on the nucleotide chain without disturbing the Watson–Crick base-pairing. We show that 5’-C bromo or propargyl convertible nucleotides (CvN) are accessible in pure diastereoisomeric form, either for nucleophilic displacement or for CuAAC conjugation. Alternatively, the 5’-carbon can be connected in a stereo-controlled manner to the phosphate moiety of the nucleotide chain to generate conformationally constrained nucleotides (CNA). These allow the precise control of the sugar/phosphate backbone torsional angles. The consequent modulation of the nucleic acid shape induces outstanding stabilization properties of duplex or hairpin structures in accordance with the preorganization concept. Some biological applications of these distorted oligonucleotides are also described. Effectively, the convertible and the constrained approaches have been merged to create constrained and convertible nucleotides (C₂NA) providing unique tools to functionalize and stabilize nucleic acids.     

Internal derivatization of oligonucleotides with selenium for X-ray crystallography using MAD.

We have developed a route for the synthesis of 2'-selenium uridine analogues and oligonucleotides containing selenium labels, and have demonstrated for the first time a new strategy to covalently derivatize nucleotides with selenium for phase and structure determination in X-ray crystallography.     

Template-free segmental synthesis of oligonucleotides containing nonnative linkages

Protected oligonucleotides containing 3'-alkyl carboxylic acids or 3'-alkylamines were obtained from photolabile solid-phase synthesis supports (1 and 4). Protected oligonucleotides containing 5'-alkylamines and 3'-hydroxyl groups were obtained using a photolabile solid-phase synthesis support (2) and a commercially available phosphoramidite reagent (3). Depending upon the source of alkylamine-containing oligonucleotide, the segments were coupled under mild conditions to form products containing either 5'-3' or 3'-3' linkages in good yield and high purity. Oligonucleotides as long as 40 nucleotides were prepared, and coupling yields of protected biopolymer segments were independent of length over the range examined. This method is particularly well suited for the convergent synthesis of oligonucleotides containing nonnative linkages and should be useful for the rapid assembly of modified biopolymers that are useful in biochemical studies.     

Bismethylene triphosphate nucleotides of uridine 4-phosphate analogues: a new class of anionic pyrimidine nucleotide analogues

Cytidine-5'-triphosphate synthase (CTPS) catalyzes the formation of cytidine triphosphate (CTP) from glutamine, uridine 5'-triphosphate (UTP), and adenosine 5'-triphosphate (ATP). This reaction proceeds via formation of the high-energy intermediate UTP-4-phosphate (UTP-4-P). Stable analogues of UTP-4-P may be potent inhibitors of CTPS and useful as lead structures for the development of anticancer and antiviral agents. Several bismethylene triphosphate (BMT) nucleotides of uridine 4-phosphate (U-4-P) analogues have been prepared. A key step was the selective methanolysis, with the aid of a tin catalyst, of the 5' ester moiety of 2',3',5'-tri-O-acetyl or tri-O-benzoyl U-4-P analogues. We believe this represents the first general approach to the selective cleavage of 5' benzoyl esters in benzoylated nucleosides. Mitsunobu coupling of these 5'-deprotected U-4-P analogues to an unsymmetrical, protected BMT bearing a free phosphonic acid moiety at one of the terminal positions gave fully protected BMT-U-4-P analogues. Global deprotection of these species was achieved using TMSBr followed by treatment with NH4OH-MeOH or NH4OH-pyridine. The resulting BMT nucleotides represent a new class of anionic pyrimidine nucleotide analogues.     

Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA.

Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.     

One-step, regioselective synthesis of up to 50-mers of RNA oligomers by montmorillonite catalysis

5'-Nucleotides of A and U with the phosphate activated with 1-methyladenine generate RNA oligomers containing 40-50 monomers in 1 day in reactions catalyzed by montmorillonite. The corresponding monomers of C give oligomers that are 20-25-mers in length after a 9-day reaction. It was not possible to determine the chain lengths of the oligomers of G since they did not give well-defined bands on gel electrophoresis. Co-oligomers of A and U as well as A, U, G, and C were also prepared. The oligo(A)s formed were separated by gel electrophoresis, and the bands of the 7-39-mers were isolated, the 3',5'-phosphodiester bonds were cleaved by RNase T(2), and the terminal phosphate groups were cleaved with alkaline phosphatase. HPLC analysis revealed that the proportions of A(5)'pp(5)'A, A, A(2)'pA, and A(2)'pA(2)'pA formed were almost the same for the long and shorter oligomers. A similar structure analysis performed on the oligo(U)s established that the proportions of U(5)'pp(5)'U, U, U(2)'pU, U(2)'pU(2)'pU, U(2)'pU(2)'pU(2)'pU, and U(2)'pU(2)'pU(2)'pU(2)'pU did not vary with chain length. The structural analysis of the oligomers of A revealed that 74% of the phosphodiester bonds were 3',5'-linked a value slightly greater than 67% observed when imidazole was the activating group. 61% of the bonds in the U oligomers were 3',5'-linked, which is almost 3 times greater than the 20% measured when imidazole was the activating group. The potential significance of these data to the origin and early evolution of life is discussed.