New Approach to the Solid Phase Synthesis of N3′→P5′ Phosphoramidate Oligonucleotides

Abstract

LCA-CPG-nucleoside 5′-O-(O-β-cyanoethyl-H-phosphonates) react with 3′-amino-2′,3′-dideoxynucleoside in the presence of iodine giving in a high yield N3′→P5′ phosphoramidate oligonucleotides.     

Protein and RNA of Human Telomerase as Targets for Modified Oligonucleotides

Abstract

Chimeric oligodeoxynucleotides containing phosphorothioate and N3′→P5′phosphoramidate linkages were synthesized. These oligomers show a high inhibitory activity against human telomerase.     

A Remarkable Stabilization of Complexes Formed by 2,6-Diaminopurine Oligonucleotide N3′→P5′ Phosphoramidates

Abstract

2′-Deoxyribo- and ribo-oligonucleotide N3′→P5′phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 °C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3′→5′ phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.     

Thermodynamic and NMR Study of Proton Complex Formation of 2′-Deoxyadenylyl-(3′→5′)-2′-Deoxyadenosine in Aqueous Solution

Abstract

A thermodynamic and proton NMR study has been carried out in order to characterize the behaviour of 2′-deoxyadenylyl-(3′→5′)-2′-deoxyadenosine towards the protonation. Conformational changes occurring following two protonation steps are discussed.     

Synthesis and in vitro activities of new anticancer duplex drugs linking 2′-deoxy-5-fluorouridine (5-FdU) with 3′-C-ethynylcytidine (ECyd) via a phosphodiester bonding

Two isomeric cytostatic duplex drugs 2′-deoxy-5-fluorouridylyl-(3′→5′)-3′-C-ethynylcytidine [5-FdU(3′→5′)ECyd] and 2′-deoxy-5-fluorouridylyl-(5′→5′)-3′-C-ethynylcytidine [5-FdU(5′→5′)ECyd] were designed and synthesized at gram scale according to the hydrogenphosphonate method in an overall yield of about 40%. The in vitro evaluation of the anticancer effects indicated highly varying sensibilities of the panel of 60 tested tumor cell lines against the duplex drugs. 5-FdU(3′→5′)ECyd had a 50% growth inhibition (IC₅₀ ? 10^?8 M) in 44/58 cell lines. However, only 25/53 of those cell lines showed corresponding IC₅₀ values when the isomeric 5-FdU(5′→5′)ECyd was tested. Total growth inhibition was achieved using micromolar concentrations of the duplex drugs. The 5-FdU residue of the duplex drug can cause very different effects like additive, synergistic, antagonistic as well as sequence-depending activities, which drastically changed efficiency as well as specificity of the anticancer activities of the duplex drugs, in comparison to those of the monomeric drugs.     

Excision of gamma-ray damaged thymine by E. coli extracts is due to the 5'leads to 3' exonuclease associated with DNA polymerase I

Extracts of $\text{E. coli pol}$Aexl which contains a temperature sensitive 5′→3′ exonuclease function of polymerase I accomplish the selective excision of products of the 5,6-dihydroxy-dihydrothymine type from γ-irradiated DNA and OsO₄-oxidized polyd(A-T) at the permissive temperature (30°) but not at the nonpermissive temperature (42°). The 5′→3′ exonuclease activity of polymerase I, therefore, acts as a repair exonuclease in γ-ray excision repair.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

Existence of Sixteen 2′→5′ Dinucleotides in Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Sixteen 2′→5′ dinucleotides; (2′–5′)pA-A, pA-G, pA-C, pA-U, pG-A, pG-G, pG-C, pG-U, pC-A, pC-G, pC-C, pC-U, pU-A, pU-G, pU-C, and pU-U were detected in nuclease P₁ digest of a technical grade yeast RNA by means of gel filtration on Sephadex G-10, DEAE-Sephadex A-25 column chromatography in the presence of 7 m urea, paper electrophoresis and paper chromatography. Content of each dinucleotide was about 0.1 to 0.6% of the digest. As the sixteen 2′→5′ dinucleotides were found in all of the digests of technical grade RNA preparations tested, each polynucleotide chain in the preparations may be concluded to contain several per cent of the 2′–5′ minor phosphodiester linkages in addition to the 3′–5′ major phosphodiester linkages.     

Studies on Synthesis and Structure of O-β-D-Ribofuranosyl(1″→2′)-ribonucleosides and Oligonucleotides♣

Abstract

Minor nucleosides found in several eukaryotic initiator tRNAs_i ^Met, O-β-D-ribofuranosyl(1″→2′)adenosine and -guanosine (Ar and Gr), as well as their pyrimidine analogues, were obtained from N-protected 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)ribonucleosides and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence of tin tetrachloride in 1,2-dichloroethane. A crystal structure has been solved for 2′-O-ribosyluridine. The 3′-phosphoramidites of protected 2′-O-ribosylribonucleosides were prepared as the reagents for 2′-O-ribofuranosyloligonucleotides synthesis. O-β-D-Ribofuranosyl(1″→2′)adenylyl(3′→5′)guanosine (ArpG) was obtained and its structure was analysed by NMR spectroscopy.

     

A Molecular Dynamics Computer Simulation Study of Nucleotide Analogues. Comparison of the Hydration Pattern of Dithymidine Phosphate with those of the Dithymidine Methylphosphonate Diastereomers

Abstract

The hydration pattern of thymidyl(3′→5′) thymidine 1 and those of R_p and S_p diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between R_p and S_p diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5′-yl units.     

Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates,and cooperates with polynucleotide phosphorylase in 3′ end maturation

Ribonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3′→5′ exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non‐viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two‐step maturation of mRNA 3′ ends, with RNR1 polishing 3′ termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non‐polysomal fractions, and half‐life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3′→5′ exoribonucleases in chloroplast mRNA 3′ end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes.     

Phenolic compounds from Selaginella moellendorfii

Chemical investigation of the leaves and roots of Selaginella moellendorfii Hieron has resulted in the isolation and characterization of two new flavone glucosides, 7‐O‐(β‐glucopyranosyl(1→2)‐[β‐glucopyranosyl(1→6)]‐β‐glucopyranosyl)flavone‐3′,4′,5,7‐tetraol ( 1 ) and 7‐O‐(β‐glucopyranosyl(1→2)‐[β‐glucopyranosyl(1→6)]‐β‐glucopyranosyl)flavone‐4′,5,7‐triol ( 2 ), two new biflavonoids, 2,3‐dihydroflavone‐5,7,4′‐triol‐(3′→8″)‐flavone‐5″,6″,7″,4′′′‐tetraol ( 3 ) and 6‐methylflavone‐5,7,4′‐triol‐(3′→O→4′′′)‐6″‐methylflavone‐5″,7″‐diol ( 4 ), two new lignans, (7′E)‐3,5,3′,5′‐tetramethoxy‐8 : 4′‐oxyneolign‐7′‐ene‐4,9,9′‐triol ( 5 ) and 3,3′‐dimethoxylign‐8′‐ene‐4,4′,9‐triol ( 6 ), together with two known monolignans, four known lignans, and four known biflavonoids. Their structures were established by spectroscopic means and by comparison with literature values.     

Transcriptional role in DNA replication: degradation of RNA primer during DNA synthesis

DNA synthesis catalyzed in vitro by E. coli DNA polymeraseI in the presence of single stranded fd DNA or poly (dT) as template is stimulated by RNA primers. When poly(dT) fully or partially saturated with polyriboadenylic acid strands is used as template - primer, DNA synthesis proceeds with concomitant degradation of the ribostrands to 5′-adenosine monophosphate. The fragment of DNA polymerase lacking the 5′→3′ exonuclease shows comparable RNA primer dependency but reduced efficiency for the degradation of the RNA primer from the 5′-end.     

Étude par r.m.n. de quatre disaccharides peracétylés sélectivement enrichis en 13C

Four peracetylated disaccharides ¹³C-labelled at the C-1′ position and having α-d-(1′→3), β-d-(1′→3), α-d-(1′→4), and β-d-(1′→4) linkages were prepared starting from the commercially available d-[1-¹³C]glucose. They were studied on the basis of their ³J_¹³CH coupling constants in relation with the conformation in solution of oligosaccharides as models for the corresponding polymer. A method of analysis of the n.m.r. spectra is described and the coupling constants J_{¹³C-1′H} given, particularly the ²J coupling (in the same cycle and with sign determination) and the ³J coupling (through the glycosidic bond). In that case, the values obtained give experimental information on the ψ angle values. They are compared with the known X-ray data for similar compounds.     

Synthesis and Enzymatic Deprotection of Fully Protected 2′‐5′ Oligoadenylates (2‐5A): Towards a Prodrug Strategy for Short 2‐5A

Fully protected pA2′p5′A2′p5′A trimers 1a and 1b have been prepared as prodrug candidates for a short 2′‐5′ oligoadenylate, 2‐5A, and its 3′‐O‐Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)‐triggered deprotection in HEPES buffer (pH 7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2‐5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2‐5A trimer, although partial removal of the 3′‐O‐[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2′,5′→3′,5′ phosphate migration and release of adenosine as side reactions.     

Synthesis of Guanylyl (3′→5′)-5-methylcytidine (Gpm5C), a Dinucleoside Monophosphate which is Unable to Function as a Primer in the Synthesis of RNA by the Influenza A Virus RNA Polymerase

Abstract

Guanylyl (3′→5′)-5-methylcytidine (Gpm⁵C) has been synthesized enzymatically through the use of T₁RNAse at high enzyme dilution. In contrast with GpC, the methylated dinucleoside monophosphate is shown to be inactive as a primer for RNA synthesis by the RNA-dependent RNA polymerase of Influenza A virus.     

The effect of ionising radiation and chemical methylation upon the activity and accuracy of E. COLI DNA polymerase I

The activity of $\text{E. coli}$ DNA polymerase I decreases on treatment with γ-rays, methylnitrosourea or dimethyl sulphate. In the case of the first two agents the decrease in activity is accompanied by a decrease in the accuracy of the enzyme in an $\text{in vitro}$ assay. There is no detectable change in the ratio of DNA polymerase activity to 3′→5′ exonuclease activity on treatment.