Steric Effect of a Bulky Substituent At 5′-Position on the Regioselectivity of 2′ vs. 3′-O-Methylation of N6- Cyclohexyladenosine

Abstract

Steric effect of a trityl or substituted trityl group at 5′-OH of N⁶-cyclohexyladenosine on the regioselective 2′ vs. 3′-O-methylation under phase transfer catalysis conditions was investigated. Compound 4 showed only a modest increase in the selectivity of 2′ over 3′-O-methylation compared to compound 1.     

Structurally Altered Substrates for DNA Topoisomerase. I. Effects of Inclusion of a Single 3′-Deoxynucleotide Within the Scissile Strand†

Abstract

A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3′-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5′-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme.

     

Study on Disulfur-Backboned Nucleic Acids: Part 3. Efficient Synthesis of 3′,5′-Dithio-2′-Deoxyuridine and Deoxycytidine

A general method is described for synthesizing 3′,5′-dithio-2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′-deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl-3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH^? ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed.     

Effect of 2-Amino Substitution on the Antiviral Effects of 5-Ethyl-2′-Deoxyuridine and (E)-5-(2-bromovinyl)-2′-Deoxyuridine and Their Incorporation into DNA

Abstract

The 2-amino derivatives of 5-ethyl-2′-deoxyuridine (EDU) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) have been synthesized and evaluated for anti-herpesvirus activity. They were at least 1000-fold less effective against herpes simplex virus replication than the parent compounds EDU and BVDU. The 5′-triphosphates of the 2-amino substituted EDU, BVDU and thymidine derivatives were also synthesized and examined on their substrate/inhibitor properties against different DNA polymerases. None of the compounds proved markedly inhibitory to HSV-1 DNA polymerase or cellular DNA polymerase a. Nor were they incorporated into the growing DNA chain.     

Biologically Active Oligodeoxyribonucleotides. VII. Anti-HIV-1 Activity of Hexadeoxyribonucleotides Bearing 3′- and 5′-End-Modifications

Abstract

It has been determined that hexadeoxyribonucleotides (5′TGGGAG3′), which have modified aromatic groups such as the trityl group at the 5′-end, exhibit anti-HIV-1 activity in vitro. The 6-mer (S-1443) bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end and a 2-hydroxyethylphosphate group at the 3′end exhibited the most potent activity and the least cytotoxicity. Moreover, it was found that the S-1443 was the most stable, when the 6-mer analogues were incubated with mouse, rat, or human plasma.     

Studies of DNA dumbbells. VI. Analysis of optical melting curves of dumbbells with a sixteen-base pair duplex stem and end-loops of variable size and sequence

Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5′-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3′ linked on both ends by single-strand loops of A_t or C_t sequences (˛ = 2, 3, 4, 6, 8, 10, 14), T_t sequences (˛ = 2, 3, 4, 6, 8, 10), and G_t sequences (t = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM Na⁺. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation ΔG_loop(n > t) and single-strand circles, ΔG_cir(N) as a function of end-loop size, t = 2-14, circle size, N = 32 + 2_t, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy ΔH, entropy ΔS, and free energy ΔG, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na⁺]. Since the multistate analysis is based on the numerically exact model, and considers a statistically significant number of theoretically possible partially melted states, it does not require prior assumptions regarding the nature of the melting transition, i.e., whether or not it occurs in a two-state manner. For comparison with the multistate analysis, thermodynamic transition parameters were also evaluated directly from experimental melting curves assuming a two-state transition and using the graphical van't Hoff analysis. Comparisons between results of the multistate and two-state analyses suggested dumbbells with loops comprised of six or fewer residues melted in a two-state manner, while the melting processes for dumbbells with larger end-loops deviate from two-state behavior.Dependence of thermodynamic parameters on[Na⁺] as a function of loop size suggests single-strand end-loops have different counterion binding properties than the melted circle. Results are compared with those obtained in an earlier study of dumbbells with the slightly different stem sequence 5'-G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C-3' linked on the ends by T loops (˛ = 2,3,4,6,8,10,14).© 1996 John Wiley &Sons, Inc.     

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs for the Formation of Duplex and Triplex DNA

(1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane (10) and their analogs were synthesized, incorporated into the oligodeoxynucleotides, and their properties were evaluated for the formation of duplex and triplex DNA. The known chiral cyclopentanone derivative was converted into the corresponding ketimine sulfonamide derivative, which was subjected to a stereoselective PhLi addition. The formed sulfonamide was hydrolyzed to afford the primary amino group, on which the thymine moiety was built. The benzyl protecting groups were removed to form the nucleoside analog having a phenyl group and the thymine unit at the 1′ position of a carbocyclic skeleton (10). In the estimation of the oligodeoxynucleotides incorporating 10 for duplex and triplex formation, the carbocyclic nucleoside analog 10 did not show the stabilizing effect for duplex formation; on the other hand, it stabilized the triplex. Therefore, the skeleton of the phenyl-substituted carbocyclic nucleoside analog 10 may be a platform for the formation of stable triplex DNA.     

5′-Phosphonates of Ribonucleosides and 2′-Deoxyribonucleosides: Synthesis and Antiviral Activity

Abstract

5′-Phosphonates of natural 2′-deoxynucleosides and ribonucleosides were synthesized by condensation of 3′-O-acylated 2′-deoxynucleosides or 2′,3′-substituted (2′,3′-O-isopropylidene, 2′,3′-O-methoxymethylene or 2′,3′-O-ethoxymethylene) ribonucleosides. As condensing agents, either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulphonyl chloride were used. Nucleoside 5′-ethoxycarbonylphosphonates were converted into corresponding nucleoside 5′-aminocarbonylphosphonates by action of ammonia in methanol or aqueous ammonia. 5′-Hydrogenphosphonothioates of thymidine and 3′-deoxythymidine were obtained by reaction of phosphinic acid in the presence of pivaloyl chloride with 3′-O-acetylthymidine or 3′-deoxythymidine, respectively, followed by addition of powedered sulfur. 5′-O-methylenephosphonates of thymidine and 2′-deoxyadenosine were prepared by intramolecular reaction of corresponding 3′-O-iodomethylphosphonates under basic conditions. All compounds were tested for inhibition of several viruses, including HSV-2 and CMV, but showed no activity. A few compounds insignificantly inhibited HIV-1 reproduction. Thymidine 5′-hydrogenphosphonate neutralized anti-HIV action of 3′-azido-3′-deoxythymidine (AZT) and it indirectly showed that even some nucleoside 5′-phosphonates could be partly hydrolyzed in cell culture to corresponding nucleosides.

5′-Phosphonates of modified 2′-deoxynucleosides in which one group in a phosphate residue is substituted for hydrogen, alkyl or other groups, have shown to be potent biologically     

Reasons and Limits of Substrate Activity of Modified L-dNTP in DNA Biosynthesis

Abstract

Theoretical and experimental analysis of interaction of modified D- and L- dNTP as substrates for template-dependent and template-independent DNA polymerases was performed. It is shown that if the modified nucleoside 5′-triphosphates do not contain a substituent in position 3′ DNA chains can be extended by both strereoisomeric series with the same kinetic parameters. But the presence of even a 3′- hydroxy group in L-dNTP prevents their incorporation into the DNA chain.     

Endcaps for Stabilizing Short DNA Duplexes

Abstract

The syntheses of endcaps for covalently linking the 3′ and 5′ hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by T_m experiments on the self-complementary octamer.     

Explicit Solvent Molecular Dynamics Simulation of Duplex Formed by the Modified Oligonucleotide with Alternating Phosphate/Phosphonate Internucleoside Linkages and its Natural Counterpart

Abstract

Impact of the internucleoside linkage modification by inserting a methylene group on the ability of the modified oligonucleotide to hybridize with a natural DNA strand was studied by fully solvated molecular dynamics (MD) simulations. Three undecamer complexes were analyzed: natural dT₁₁.dA₁₁ duplex as a reference and two its analogs with alternating modified and natural linkages in the deoxyadenosine chain. The isopolar, non-isosteric modified linkages were of 5′-O-PO₂-CH₂-O-3′ (5′PC3′) or 5′-O-CH₂-PO₂-O-3′ (5′CP3′) type. Simulations were performed by using the AMBER 5.0 software package with the force field completed by a set of parameters needed to model the modified segments. Both modifications were found to lead to double helical complexes, in which the thymidine strand as well as deoxyriboses and unmodified linkages in the adenosine strand adopted conformations typical for the B-type structure. For each of the two conformational richer modified linkages two stable conformations were found at 300 K: the -ggt and ggt for the 5′PC3′ and ggg, tgg for the 5′CP3′, respectively. Both modified chains adopted helical conformations with heightened values of the inclination parameter but without affecting the Watson-Crick hydrogen bonds.     

Synthesis and Biological Activities of Some New 5′-Substituted S-Adenosylhomocysteine Analogues∗

Abstract

The synthesis of new S-adenosyl-homocysteine (SAH) analogues substituted at the 5′-position is described. Their activity on cell transformation induced by Rous sarcoma virus (RSV) as well as their effect on cell growth and on replication of polyoma virus were also studied. The best results were obtained with compounds having a phenyl group at the 5′-position of adenosine. These molecules inhibit very strongly uridine uptake by cells.     

Synthesis of 8-Bromo- and 8-Azido-2′-deoxyadenosine-5′-O-(1-thiotriphosphate)

Abstract

Treatment of 3′-O-methoxyacetylated 8-bromo-2′-deoxyadenosine (5), with a twofold excess of salicyl phosphorochloridite (6), and subsequent reaction with bis(tri-n-butylammonium) pyrophosphate and oxidation with sulfur followed by removal of the protecting group gives predominantly 8-bromo-2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (7), and minor amounts of the corresponding brominated monothiophosphate. Alternatively, the photoreactive dATP analog 8-azido-2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (11), is obtained by phosphorylation of unprotected 8-azido-2′-deoxyadenosine (9) with a 1.8 molar equivalent excess of thiophosphoryl chloride and bis(tri-n-butylammonium) pyrophosphate. A protection of the nucleobase 6-amino group is not required. The photoaffinity labeling reagent 11, was characterized by ³¹P-NMR and ion-spray mass spectroscopy and its photolysis upon long wavelength UV irradiation was studied. Both α-thioderivatives of 2′-deoxyadenosine triphosphates can be incorporated into plasmid DNA by T7 DNA polymerase. Thus, they can be used for interference studies of protein binding and for cross-linking with amino acids in protein-nucleic acid-complexes.     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

Structure-Antibacterial Activity Relationship for 9-O,9′-O-Demethyl (+)-virgatusin

The relationship between the antibacterial activity and structure of 9-O,9′-O-demethyl (+)-virgatusin (Virg 3) was examined. The conversion of hydroxy groups on the 9 and 9′ positions to amino groups increased the activity. It was found that the 3′-methoxy group was more important for higher activity than the 4′-methoxy group on the 7′-phenyl group, and that the 3,4-methylenedioxy group on the 7-phenyl group was necessary for activity.     

Aminoacyl-tRNA Analogues; Synthesis,Purification and Properties of 3′-Anthraniloyl Oligoribonucleotides

Abstract

Reaction of isatoic anhydride with adenosine, adenosine 5′-phosphate, oligoribonucleotides or with the E. coli tRNA^Val led to attachment of an anthraniloyl residue at 2′-or 3′-OH groups of 3′-terminal ribose residue. No protection of the S'-hydroxyl group or internal 2′-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

     

7-Deaza-2′-Deoxyxanthosine: Nucleobase Protection and Base Pairing of Oligonucleotides

Oligonucleotides containing 7-deaza-2′-deoxyxanthosine (1) and 2′-deoxyxanthosine (2) were prepared. The 2-(4-nitrophenyl)ethyl group is applicable for 7-deazaxanthine protection that is removed with DBU by β-elimination, while the deprotection of the allyl residue with Pd (0) catalyst failed. Contrarily, the allyl group was found to be an excellent protecting group for 2′-deoxyxanthosine (2). The base pairing of nucleosides 1 and 2 with the four canonical DNA constituents as well as with 3 within the 12-mer duplexes is studied.     

DNA-Triesters - The Synthesis and Absolute Configuration Assignments at P-Stereogenic Centres

Abstract

Decadeoxyribonucleotide GGGAATTCCC and nine diastereomeric pairs of its mono-O-ethyl ester analogues were synthesized via phosphoramidite approach using the combination of 5′-DMT-base protected (except T) nucleoside 3′-(2-cyanoethyl N,N-diisopropyl phosphoramidites) and 3′-(0-ethyl N,N-diisopropyl phosphoramidites). Under conditions of release from solid support and removal of base-protecting groups (25% NH₄OH, 25°C, 48 h) 2-cyanoethyl groups were removed while O-ethyl phosphate triester functions were practically intact. Isolation of products and separation of diastereomers were performed by means of RP-HPLC. Absolute configuration at P-stereogenic centres was established via degradation of decamers into corresponding dinucleoside O-ethyl phosphates and stereochemical correlation with dinucleoside phosphorothioates of known configuration at phosphorus. Decadeoxyribonucleotide mono-O-ethyl esters were used for mapping the contact points between DNA and Eco RI endonuclease - the restriction enzyme which recognizes canonical sequence. GAATTC and cleaves unmodified DNA strands giving G and p AATTC.     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.