Inhibition of duck hepatitis B virus replication by purine 2',3'-dideoxynucleosides

Primary hepatocyte cultures from duck hepatitis B virus (DHBV) infected ducklings were used to evaluate the antiviral activity of purine and pyrimidine 2',3'-dideoxynucleosides. The purine 2',3'-dideoxynucleosides were very effective inhibitors of hepadnavirus replication, whereas the pyrimidine dideoxynucleosides were not. 2',3'-Dideoxyguanosine and 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR) were the most effective antiviral agents studied. ddDAPR given intramuscularly twice daily at 10 mg/kg rapidly cleared DHBV-DNA from the sera of persistently infected ducklings but this effect was not permanent.     

2',3'-Dideoxynucleoside phosphorylation by deoxycytidine kinase from normal human thymus extracts: activation of potential drugs for AIDS therapy

As a first step toward improving dideoxynucleoside inhibition of human immunodeficiency virus replication in human lymphocytes, we examined the kinetics of 5'-phosphorylation of a series of 2',3'-dideoxynucleosides, using deoxycytidine kinase purified from human thymus extracts. Nucleosides with the 2'-deoxyribose moiety were activated 30 times faster than were 2',3'-dideoxynucleosides. The adenosine deaminase inhibitor, 2'-deoxycoformycin, showed an unexpected ability to inhibit purine and pyrimidine dideoxynucleoside phosphorylation; such inhibition was not competitive and was not observed when 2'-deoxycytidine was the substrate. 2'-Deoxycytidine, the natural substrate, inhibited dideoxynucleoside phosphorylation in a manner similar to that observed with 2'-deoxycoformycin. Thus, dideoxynucleosides are activated by deoxycytidine kinase through a different catalytic interaction than occurs in 5'-activation of 3'-hydroxynucleosides by this enzyme.     

Estimation of the lipophilicity of anti-HIV nucleoside analogues by determination of the partition coefficient and retention time on a Lichrospher 60 RP-8 HPLC column

There is a close linear correlation between the log partition coefficient (Pa) of a series of 2'-deoxyriboside (dR), 2',3'-didehydro-3'-dideoxyriboside (ddeR), 2',3'-dideoxyriboside (ddR), 3'-fluoro-2',3'-dideoxyriboside (FddR) and 3'-azido-2',3'-dideoxyriboside (AzddR) derivatives of uracil, cytosine, thymine, guanine, adenine and 2,6-diaminopurine and their retention times (Rt) on a Lichrospher 60 RP-8 HPLC column (correlation coefficient r greater than 0.970). Within each class of compounds the following order of increasing lipophilicity was noted: dR less than ddeR less than ddR less than FddR less than AzddR. A straight-forward structure-lipophilicity relationship for both base and sugar modified purine and pyrimidine 2',3'-dideoxynucleosides could be delineated.     

Reactivity models of 1-N-vinyluracil and synthesis of a new class of potential antiviral agents by the use of 1,3-dipolar cycloaddition reactions

By the use of a convergent approach based on 1,3-dipolar cycloaddition reactions between N-protected formylnitrones generated in situ and 1-N-vinyluracil, a new class of 4'-aza-analogues of 2',3'-dideoxynucleosides is synthesized. Competitive reaction for the endocyclic bond of uracil also brings to a new isoxazolidine derivative fused with the pyrimidine nucleus.     

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

Local sequence dependence of rate of base replacement in mammals.

I have analysed the local sequence context of base replacement changes in 78 processed pseudogenes. Transversions occur more often than transitions in a ratio of 3.37 to 1, and G:C is replaced 1.4 times more frequently than A:T. In addition, the bases to the 5' and 3' of the mutating base also influence the rate at which bases change, purine:pyrimidine and pyrimidine:purine pairs changing 1.2 times as fast as purine:purine and pyrimidine:pyrimidine pairs. I discuss implications of this for the mechanism of DNA polymerization in mammals.     

Molecular mechanics calculations on a triple stranded DNA involving C+.G-T and T.A+-C mismatched base triples

We have carried out molecular modeling of a triple stranded pyrimidine(Y). purine(R): pyrimidine(Y) (where ':' refers to Watson-Crick and '.' to Hoogsteen bonding) DNA, formed by a homopurine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC). Molecular mechanics calculations using NMR constraints have provided a detailed three dimensional structure of the triplex. The entire stretches of purine and the pyrimidine nucleotides have a conformation close to B-DNA. The three strands are held by the canonical C+.G:C and T.A:T hydrogen bonds. The structure also contains two mismatch C+.G-T and T.A+-C base triples which have been characterized for the first time. In the A+-C base-pair of the T.A+-C triple, both hydrogen donors are situated on the purine (A+(1N) and A+(6N)). We observe a unique hydrogen bonding interaction scheme in case of C+.G-T where one acceptor, G(60), is bonded to three donors (C+(3NH), C+(4NH2) and T(3NH)). Though the C+.G-T base triple is less stable than C+.G:C, it is significantly more stable than T.A:T. On the other hand, T.A+-C is as stable as the T.A:T base triad.     

Alkaline ribonuclease from rye germ cytosol.

1. Alkaline ribonuclease (pH optimum 7.6) was isolated from rye (Secale cereale L) germ cytosol and partially purified; the preparation was devoid of other nucleolytic activities. 2. The enzyme is a typical endonuclease hydrolysing all phosphodiester bonds in RNA, yielding ultimately purine and pyrimidine nucleoside 2',3'-cyclic phosphates and the corresponding 3'-phosphates. Upon extensive digestion of synthetic polyribonucleotides, pyrimidine, but not purine, nucleoside 3'-phosphates are formed. The enzyme does not hydrolyse synthetic purine cyclic nucleotides. 3. The enzyme does not depolymerize double-stranded complexes of poly(A) and poly(U). 4. Susceptibility to photooxidation and inhibition by 2-hydroxy-5-nitrobenzyl bromide and N-bromosuccinimide implies the involvement of tryptophan residue in the active centre of the enzyme.     

Nucleobase transporter-mediated permeation of 2',3'-dideoxyguanosine in human erythrocytes and human T-lymphoblastoid CCRF-CEM cells.

Several 2',3'-dideoxynucleosides (ddNs), agents that inhibit the replication of human immunodeficiency virus and hepatitis B virus, enter mammalian cells by simple diffusion. In this report, we show that the membrane permeation of 2',3'-dideoxyguanosine (ddG) in human erythrocytes and CCRF-CEM cells, in contrast with that of other ddNs, is transporter-mediated. Inward fluxes of ddG in both cell types were inhibited by adenine, hypoxanthine, and acyclovir, but not by inhibitors of nucleoside transport (nitrobenzylthioinosine, dipyridamole, dilazep). Fluxes of ddG in human erythrocytes were attributable to a single, rate-saturable process (Km, 380 +/- 90 microM and Vmax, 7.9 +/- 0.8 pmol/s/microliter cell water) that was competitively inhibited by adenine (Ki, 16 microM). These results showed that ddG entered human erythrocytes and CCRF-CEM cells by a transporter-mediated process that was also the basis for entry of purine nucleobases. In contrast, inward fluxes of 2,6-diaminopurine-2',3'-dideoxyriboside (ddDAPR), a prodrug of ddG, were not affected by purine nucleobases or nucleoside transport inhibitors in either cell type. Thus, the permeation properties of ddDAPR resembled those of 2',3'-dideoxyadenosine, a diffusional permeant (cell uptake is transporter-independent), and contrasted with those of ddG, the deamination product of ddDAPR. This study demonstrated that the nucleobase moiety of ddNs is an important determinant of membrane permeation.     

Inhibition of various stages of DNA replication in sea urchin embryos

The embryos of the sea urchin Strongylocentrotus intermedius possess the ability to incorporate into their DNAs 2'-deoxynucleosides together with all their bases, i.e., adenine, guanine, cytosine and thymine. This incorporation is inhibited by 3'-amino-2',3'-dideoxynucleosides with the same bases. 5'-Amino-5'-deoxynucleosides and 5'-amino-2',5'-dideoxynucleosides moderately inhibit the incorporation of [3H]2'-deoxynucleosides into the DNAs by competing with the latter, presumably at the phosphorylation stage. The most potent inhibiting effect is exerted by 2'-amino-2'-deoxynucleosides and 2'-asido-2'-deoxynucleosides; the mechanism of this inhibition is still obscure, however.     

Synthesis of 2',3'-dideoxy-3'-fluoro-L-ribonucleosides as potential antiviral agents from D-sorbitol

2',3'-Dideoxy-3'-fluoro-L-ribonucleosides were synthesized as potential antiviral agents. The key intermediate, methyl 5-O-benzoyl-2,3-dideoxy-3-fluoro-L-ribofuranoside, which was prepared from D-sorbitol, was condensed with pyrimidine and purine bases to obtain the respective nucleosides. Among them, the cytosine analogue 2',3'-dideoxy-3'-fluoro-alpha-L-cytidine showed a moderate anti-HBV activity.     

A theoretical investigation on the sequence selective binding of mitoxantrone to double-stranded tetranucleotides.

Theoretical computations are performed on the comparative binding energetics of mitoxantrone (MX), a newly synthesized intercalating anthraquinone antitumor drug, to six representative double-stranded tetranucleotides: d(GCGC)2, d(CGCG)2, d(ATAT)2, d(TATA)2, d(GTGT), d(ACAC), and d(CCGG)2. The computations are performed with the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. The best binding configuration of mitoxantrone locates its two side chains in the major groove. A considerable preference is elicited for intercalation of the chromophore ring in a pyrimidine (3'-5') purine sequence rather than the isomeric purine (3'-5') pyrimidine sequence. Contrary to the situation encountered with "simple" intercalators, in which this preference is generally attributed solely to differences in the energies of unstacking necessary to generate the intercalation sites, the preference is dictated in MX to a large extent by the intermolecular interaction energy term. This result is imposed by the interactions of the side chains of MX with the oligonucleotide.     

Synthesis and anti-HIV activity of beta-D-3'-azido-2',3'-unsaturated nucleosides and beta-D-3'-azido-3'-deoxyribofuranosylnucleosides

Since the discovery of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2',3'-didehydro-2',3'dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Production of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine from 2',3'-dideoxyuridine and the corresponding purine bases by resting cells of Escherichia coli AJ 2595.

A novel microbial method for the production of 2',3'-dideoxynucleosides by transdideoxyribosylation has been developed. By screening microorganisms producing 2',3'-dideoxyadenosine (DDA) from 2',3'-dideoxyuridine (DDU) and adenine, Escherichia coli AJ 2595 was selected as the best producer. Optimal pH and temperature for the DDA-producing reaction were ca. 6.5 and 50 degrees C, respectively. Pi seemed to be an essential factor for the reaction, and its optimal concentration was ca. 25 mM. Moreover, polyethylene glycol had a notable effect on DDA production. Under the best conditions established, 52 mM DDA was obtained from 100 mM DDU and 100 mM adenine after 48 h of incubation from resting cells of E. coli AJ 2595. This strain could also produce 2',3'-dideoxynucleosides, such as 2',3'-dideoxyinosine (DDI), 2',3'-dideoxyguanosine, and 2',3'-dideoxythymidine, from DDU and the corresponding bases. In particular, this strain could produce DDI in high yield (ca. 32 mM from 100 mM DDU and 100 mM hypoxanthine) after 24 h of incubation. However, 2',3'-dideoxycytidine was not produced from DDU and cytosine by resting cells of E. coli AJ 2595.     

Stacking-unstacking dynamics of oligodeoxynucleotide trimers

The structure and dynamics of DNA trimers are experimentally assessed using the fluorescent purine analogue 2-aminopurine (2AP), incorporating 2AP between purine and pyrimidine bases to form 5'dXp2APpY3' molecules. Circular dichroism and fluorescence quenching of the 2AP show that the bases are stacked; at the same time, fluorescence decay lifetimes are heterogeneous, indicative of conformational sampling. 2AP does not exhibit the long fluorescence decay time characteristic of the free nucleoside, suggesting that its motions in the trimers bring it into proximity of the neighboring bases, resulting in efficient charge transfer and average fluorescence lifetimes on the order of 1-2 ns.     

Preparation of 2'-deoxyribonucleosides with an identically 2H/13C-labeled sugar residue.

Thymidine with the stereoselectively 2H/13C-Labeled sugar moiety, (2'R)(5'S)-[1',2',3',4',5'-(13)C5;2',5'-(2)H2]-thymidine, was synthesized from uniformly 13C-labeled glucose, via the selectively deuterated ribose derivative prepared by the stereo-controlled deuteride transfer reactions. The labeled sugar moiety of the thymidine was then transferred to 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxyuridine, by the enzymatic transglycosylation reactions by purine and pyrimidine nucleoside phosphorylases, in good yields. Labeled 2'-deoxyuridine was chemically converted to 2'-deoxycytidine. Consequently, all of the 2'-deoxynucleosides prepared by this method has the identically labeled sugar moiety. By using DNA oligomers containing the identically labeled sugar residue for NMR studies, any possible complexity in NMR data analyses expected to be observed for DNA oligomers containing variously labeled nucleosides can be minimized.     

Sequence composition effects on the stabilities of triple helix formation by oligonucleotides containing N7-deoxyguanosine.

A nonnatural nucleoside, 7-(2-deoxy-beta-D-erythro-pento-furanosyl)-guanine (d7G), mimics protonated cytosine and specifically binds GC base pairs within a pyrimidine - purine - pyrimidine triple helix. The differences in association constants (KT) determined by quantitative footprint titration experiments at neutral pH reveal dramatic sequence composition effects on the energetics of triple helix formation by oligonucleotides containing d7G. Purine tracts of sequence composition 5'-d(AAAAAGAGAGAGAGA)-3' are bound by oligonucleotide 5'-d(TTTTT7GT7GT7GT7GT7GT)-3' three orders of magnitude less strongly than by 5'-d(TTTTTmCTmCTmCTmCTmCT)-3' (KT = 1.5 x 10(6) M(-1) and KT > or = 3 x 10(9) M(-1) respectively). Conversely, purine tracts of sequence composition 5'-d(AAAAGAAAAGGGGGGA)-3' are bound by oligonucleotide 5'-d(TTTTmCTTTT7G7G7G7G7G7GT)-3' five orders of magnitude more strongly than by 5'-d(TTTTmCTTTTmCmCmCmCmCT)-3' (KT > or = 3 x 10(9) M(-1) and KT < 5 x 10(4) M(-1) respectively). The complementary nature of d7G and mC expands the repertoire of G-rich sequences which may be targeted by triple helix formation.     

Production of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine from 2',3'-dideoxyuridine and the corresponding purine bases by resting cells of Escherichia coli AJ 2595

A novel microbial method for the production of 2',3'-dideoxynucleosides by transdideoxyribosylation has been developed. By screening microorganisms producing 2',3'-dideoxyadenosine (DDA) from 2',3'-dideoxyuridine (DDU) and adenine, Escherichia coli AJ 2595 was selected as the best producer. Optimal pH and temperature for the DDA-producing reaction were ca. 6.5 and 50 degrees C, respectively. Pi seemed to be an essential factor for the reaction, and its optimal concentration was ca. 25 mM. Moreover, polyethylene glycol had a notable effect on DDA production. Under the best conditions established, 52 mM DDA was obtained from 100 mM DDU and 100 mM adenine after 48 h of incubation from resting cells of E. coli AJ 2595. This strain could also produce 2',3'-dideoxynucleosides, such as 2',3'-dideoxyinosine (DDI), 2',3'-dideoxyguanosine, and 2',3'-dideoxythymidine, from DDU and the corresponding bases. In particular, this strain could produce DDI in high yield (ca. 32 mM from 100 mM DDU and 100 mM hypoxanthine) after 24 h of incubation. However, 2',3'-dideoxycytidine was not produced from DDU and cytosine by resting cells of E. coli AJ 2595.     

Three-dimensional homonuclear NOESY-TOCSY of an intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple: nonexchangeable proton assignments and structural implications.

Two- and three-dimensional homonuclear 1H NMR spectroscopic techniques have been applied to obtain nearly complete nonexchangeable proton assignments for a 31-residue intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple in D2O. An assignment strategy for obtaining resonance assignments for DNA protons from a 3D NOESY-TOCSY spectrum is proposed. The strategy utilizes the H1'/H5 omega 3 planes and relies on the recognition of cross-peak patterns for obtaining both intraresidue as well as sequential assignments. On the basis of the cross-peaks observed in the 2D and 3D spectra, a few structural features of the triplex have been delineated qualitatively. All three strands of the triplex adopt a right-handed helical conformation, and, despite the introduction of a central purine guanosine, there is no evidence for major structural distortions in the protonated third strand on the basis of a qualitative interpretation of NMR data. Several interstrand contacts between the purine and the Hoogsteen pyrimidine strands are observed which define the relative orientation of the bases and sugars in these two strands. The presence of strong NOEs between the methyl protons of thymine and the H1' proton of guanosine defines the preferred base-pairing alignment of guanosine at the G.TA triple site. The general approaches illustrated in this study extend the range of DNA molecules accessible for detailed structural investigation by high-resolution NMR spectroscopy.