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.     

Synthesis of Oligodeoxyribonucleotide Bearing 2′-S-Alkyl Residue and its Effect on the Duplex Stability

Abstract

2′-Deoxy-2′-S-hexyluridine derivative was synthesized from 2,2′-anhydrouridine and 1-hexanethiol and incorporated into an oligodeoxyribonucleotide. The thermal stability of the duplexes formed by the 2′-S-hexyl modified ODN with either the complementary DNA or RNA strand was decreased compared to the unmodified counterparts.     

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.     

Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

Highly Efficient and Facile Synthesis of 5-Azido-2′-Deoxyuridine

Previously reported syntheses of the photoaffinity label 5-azido-2′-deoxyuridine are rather inefficient and involve the tedious preparation of a 5-nitro intermediate. To overcome these inconveniences, we have developed a new approach from the commercially available 5-bromo-2′-deoxyuridine nucleoside. Our synthetic route makes use of a benzylamination reduction sequence. Using this strategy, the 5-azido-2′-deoxyuridine photolabel is prepared in three steps and quantitative yields.     

Synthesis and Antiviral Activity of New 5-Substituted 2′-Deoxyuridine Derivatives

New 5-azole- and 5-oxime-substituted analogues of 2′-deoxyuridine are synthesized. The analogues with azole ring manifest low toxicities and antiherpetic activities on Vero cell culture, the imidazole derivative being the most active. The inhibitory effects of oximes of 5-formyl-deoxyuridine are comparable with those of the azole-containing nucleoside analogues, although their cytotoxicities are found to be higher; oxime of 5-formyldeoxyuridine is particularly toxic. The nucleoside analogues synthesized exhibit no marked activity on cell cultures infected with various variants of poxvirus.     

Effects of 3′-C-Methylation on the Hydrolytic Stability and Hydroxyl pK a Values of Dinucleoside 2′,5′- and 3′,5′-Monophosphates

Abstract

The first-order rate constants for hydrolysis of 3′-C-methyluridylyl(2′,5′)- and -(3′,5′)adenosine and the corresponding native dinucleoside monophosphates (2′,5′- and 3′,5′-UpA) have been determined as a function of hydroxide-ion concentration (0.025 - 7 M) at 25°C. In addition to the effects on the hydrolytic stability of the compounds, the effects of the 3′-C-methyl substitution on the kinetically determined pK _a values for the sugar hydroxyls of the undine moiety are discussed.     

Synthesis and Biological Activity of BIS(Pivaloyloxymethyl) Ester of 2′-Azido-2′-Deoxyuridine 5′-Monophosphate

Abstract

Bis(pivaloyloxymethyl) ester of 2′-azido-2′-deoxyuridine 5′-monophosphate was prepared as a prodrug to generate 2′-azido-2′-deoxyuridine 5′-diphosphate inside the cell. A synthetic route utilizing stannyl phosphate was adopted in the preparation. The prodrug was evaluated for cell growth inhibition against a variety of tumor cell lines along with 2′-azido-2′-deoxyuridine and 2′-azido-2′-deoxycytidine.     

4′-C-Aminomethyl-2′-deoxy-2′-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting the ability of a DNA/RNA duplex to activate RNase H

An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-β-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides.     

Effect of 5′-terminal structure and base composition on polyribonucleotide binding to ribosomes

Ribopolymers of variable base composition and 5′-terminal structure were synthesized with polynucleotide phosphorylase. Under primer-dependent conditions, m⁷GpppGmpC (m⁷G-cap)², its alkali-treated m⁷G ring-opened derivative, GpppGpC and ppGpC but not m⁷GpppGmpCp, m⁷GpppGm or GpppG were incorporated as 5′-termini. The ribopolymers were compared with reovirus mRNA, which contains m⁷G-cap, for their ability to form initiation complexes with wheat germ 40 S ribosomal subunits and 80 S ribosomes. The presence of 5′-terminal m⁷G was required for stable complex formation by some ribopolymers while for others binding was increased by two- to fourfold. The final level of binding observed was similar to that with reovirus mRNA. In addition to 5′-terminal m⁷G, the base composition of the ribopolymers markedly influenced binding. Some ribopolymers including m⁷G-cap (A)_n did not bind significantly; m⁷G-cap (U)_n formed 40 S complexes while m⁷G-cap (A,U)_n bound to 80 S ribosomes. The ribopolymer m⁷G-cap (A₂,U₂,G)_n directed protein synthesis as measured by amino acid incorporation into polypeptides, methionine tRNA association with 40 S complexes, and puromycin reactivity of 80 S-associated methionine and, like reovirus mRNA, its binding to ribosomes was inhibited by 7-methylguanosine 5′-monophosphate.     

Evaluation of Different Types of End-Capping Modifications on the Stability of Oligonucleotides Toward 3′- and 5′-Exonucleases

Abstract

Synthetic oligonucleotides are increasingly used because of their potential activity as regulators of gene expression. One of their major drawbacks is instability toward nucleases, in particular exonucleases. In this article, we studied some terminal modifications that can enhance exonuclease resistance, such as end-capping with alkylic chains (1,3-propanediol and 1,6-hexanediol), and with a modified nucleotide (2′,3′ -secouridine). These compounds were compared with the parent (natural) oligodeoxynucleotide and with different analogs containing a progressive number of phosphorothioate linkages. The resistance toward SVPDE and CSPDE (a 3′ - and a 5′ -exonuclease) was assessed, in vitro, by two independent techniques, UV and HPLC. Our results showed that the stability of all the modified oligonucleotides was at least 12 times that of the parent compound.     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: II. The Synthesis of Phosphoamidites on the Basis of Uridine and Cytosine and Containing a Linker with Methoxyoxalamide Groups in Position 2′

A convenient preparative synthesis of 2-amino-2-deoxyuridine was developed. Starting from 2-amino-2-deoxyuridine and 2-amino-2-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2.     

Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases

summary. A thermostable -amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca²⁺. The stability of the native enzyme was found to increase upon modification as observed from the increase in t_1/2 in the absence of Ca²⁺ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with C_m and T_m values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.This article is dedicated to Dr. P.V. Sundaram.     

3′-O- and 5′-O-Propargyl Derivatives of 5-Fluoro-2′-Deoxyuridine: Synthesis,Cytotoxic Evaluation and Conformational Analysis

A series of new 3′-O- and 5′-O-propargyl derivatives of 5-fluoro-2′-deoxyuridine (1–4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2′-deoxyuridine analogues (1–4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3′-O-propargyl-5-fluoro-2′-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.     

Effect of single pyrrole replacement with β-alanine on DNA binding affinity and sequence specificity of hairpin pyrrole/imidazole polyamides targeting 5′-GCGC-3′

N-Methylpyrrole (Py)–N-methylimidazole (Im) polyamides are small organic molecules that can recognize predetermined DNA sequences with high sequence specificity. As many eukaryotic promoter regions contain highly GC-rich sequences, it is valuable to synthesize and characterize Py–Im polyamides that recognize GC-rich motifs. In this study, we synthesized four hairpin Py–Im polyamides 1–4, which recognize 5′-GCGC-3′ and investigated their binding behavior with surface plasmon resonance assay. Py–Im polyamides 2–4 contain two, one, and one β-alanine units, replacing the Py units of 1, respectively. The binding affinities of 2–4 to the target DNA increased 430, 390, and 610-fold, respectively, over that of 1. The association and dissociation rates of 2 to the target DNA were improved by 11 and 37-fold, respectively, compared with those of 1. Interestingly, the association and dissociation rates of 3 and 4 were higher than those of 2, even though the binding affinities of 2, 3, and 4 to the target DNA were comparable to each other. The binding affinity of 2 to DNA with a 2 bp mismatch was reduced by 29-fold, compared with that to the matched DNA. Moreover, the binding affinities of 3 and 4 to the same mismatched DNA were reduced by 270 and 110-fold, respectively, indicating that 3 and 4 have greater specificities than 2 and are suitable as DNA-binding modules for engineered epigenetic regulation.     

Stability of 5-Fluoro-2′-deoxycytidine and Tetrahydrouridine in Combination

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2′-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC®-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37°C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.     

Synthesis and Antiviral Activities of Some New 5-Heteroaromatic Substituted Derivatives of 2′-Deoxyuridine

Abstract

Eight new 5-heteroaromatic substituted analogues of 2′-deoxyuridine have been synthesized and evaluated for their inhibitory properties against a panel of different viruses. Several analogues containing a substituted thiophene moiety proved to be highly selective against herpes simplex virus type 1 (HSV-1).