Amino substituted derivatives of 5'-amino-5'-deoxy-5'-noraristeromycin

The potent antiviral potential of 5'-amino-5'-deoxy-5'-noraristeromycin (2) is limited by associated toxicity. To seek derivatives of 2 that circumvent this undesirable property, three amino substituted derivatives (acetyl, 3; formyl, 4; and methyl, 5) of 2 have been prepared in 4-7 steps from the same intermediate, (1S,4R)-4-(6-chloropurin-9-yl)cyclopent-2-en-1-ol (6). Key steps involved an improved Pd(0)-catalyzed allylic azidation and a novel Pd(0)-catalyzed allylic amidation. The three target compounds were evaluated against a large number of viruses and found to be inactive except for a very weak effect of 5 on human cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. There was also no noteworthy cytotoxicity associated with the new derivatives. Thus, these results indicate variation of the cyclopentyl amine of 2 does not offer a means to improve upon its antiviral potential.     

6-azapyrimidine and 7-deazapurine 2'-deoxy-2'-fluoroarabinonucleosides: synthesis, conformation and properties of oligonucleotides

The synthesis of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl nucleosides (1b, 2b, and 3b) were described and their conformation in solution as well as in the solid state was determined In addition to this, building blocks 10a,b and 13a,b were prepared and employed in solid-phase oligonucleotide synthesis. For compounds 1a and 1b the lactime proton is protected to avoid unresolved degradation of its phosphoramidites 10a,b. UV-melting studies have been carried out to assess the thermal stability of oligonucleotides containing compounds 1a,b, and 3a,b.     

Conformationally locked nucleosides. Synthesis of oligodeoxynucleotides containing 3'-amino-3'-deoxy-3'-N,5'(R)-C-ethylenethymidine

3'-Amino-3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-N,5'(R)-C-ethylenethymidine (6) was synthesized starting from 3'-azido-3'-deoxythymidine. Condensation of 6 with 5'O-(H-phosphonyl)thymidine and 5'-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Synthesis and properties of oligodeoxynucleotides containing the analogue 2'-deoxy-4'-thiothymidine.

The 2'-deoxythymidine analogue 2'-deoxy-4'-thiothymidine has been incorporated, using standard methodology, into a series of dodecadeoxynucleotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligodeoxynucleotides and their ability to act as substrates for the restriction endonuclease and associated methylase have been compared with a normal unmodified oligodeoxynucleotide. No problems were encountered in the synthesis despite the presence of a potentially oxidisable sulfur atom in the sugar ring. The analogue had very little effect on the melting temperature of the self-complementary oligoeoxynucleotides so synthesised and all had a CD spectrum compatible with a B-DNA structure. The oligodeoxynucleotide containing one analogue in each strand within the recognition site, adjacent to the bond to be cleaved (i.e. GAXATC, where X is 2'-deoxy-4'-thiothymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexanucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucleotide was a poor substrate for both the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the kcat value was only 0.03% of the value obtained for the parent oligodeoxynucleotide. These results show that the incorporation of 2'-deoxy-4'-thionucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2'-deoxy-4'-thiothymidine itself is so toxic in cell culture.     

Synthesis and conformation control of peptide ribonucleic acid containing 5'-amino-5'-deoxyribopurinenucleosides.

A novel nucleic acid model, i.e. peptide ribonucleic acid (PRNA), tethering 5'-amino-5'-deoxypyrimidine ribonucleoside as a recognition site for nucleic acids, has been designed and synthesized. We have demonstrated that the recognition behavior of PRNA with complementary oligopurinenucleotides can be controlled externally through the orientational switching of the pyrimidine nucleobase of PRNA induced by added borates. We extend this methodology of controlling the nucleobase orientation and recognition behavior of novel mono and oligomeric PRNAs containing 5'-amino-5'-deoxypyrimidine and/or purinenucleosides. In case of the PRNA oligomer containing pyrimidine-purine mixed sequence, efficient orientational switching of nucleobases induced by added borates was also observed.     

Synthesis and hybridization properties of oligodeoxynucleotides containing 3'-deoxy-3'-C-methyleneuridine

3'-Deoxy-3'-C-methyleneuridine nucleoside 1 has been incorporated into oligodeoxynucleotides. Relative to the unmodified references, oligomers containing nucleoside 1 displayed reduced binding affinities towards complementary DNA and RNA with a tendency towards RNA-selective hybridization.     

PNA-DNA chimeras containing 5-alkynyl-pyrimidine PNA units. Synthesis, binding properties, and enzymatic stability

Three chimeric dimer synthons (oeg_t(NH)T, oeg_up(NH)T and oeg_uh(NH)T) containing thymine (t), 5-(1-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T20 oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA20 target. The smallest Tm drops were found for chimeras containing oeg_up(NH)T dimers. Incorporation of the chimeric synthons into the 3'-end of T20 brought about growing resistance to 3'-exonucleolytic (SV PDE) cleavage in the order of oeg_t(NH)T < oeg_up(NH)T < oeg_uh(NH)T. Due to different endonuclease activities of 3'- and 5'-exonucleases applied, placing of five consecutive dimers at the 5'-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5'-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P1) could hydrolyse the unnatural phosphodiester bond linking the 3'-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.     

Synthesis and properties of 2'-deoxy-8,2'-methylene-cyclopurine nucleosides

A method was developed to synthesize 2'-deoxy-8,2'-methylene-cycloadenosine (1) and -cycloguanosine (2) which were new carbon-bridged cyclopurine nucleosides fixed in a high-anti torsional angle region. 3',5'-Di-O-acetyl-8-methanesulfonyl-2'-O-p-toluene-sulfonyladenosine+ ++ (3) or 2-acetamido-9- (3,5-di-O-acetyl-2-O-p-toluenesulfonyl-beta-D-ribofuranosyl)-6- ethoxy-8-methanesulfonyl-purine (9) was treated with sodium salt of ethyl malonate to give the cyclized nucleosides (4 and 10) in good yields, respectively. Subsequent decarboxylation and deblocking of 4 and 10 afforded 1 and 2 in crystalline form, respectively.     

Synthesis and hybridization properties of DNA-PNA chimeras carrying 5-bromouracil and 5-methylcytosine

The preparation of 5-bromouracil and 5-methylcytosine peptide nucleic acid (PNA) monomers is described. These PNA monomers were used for the preparation of several DNA-PNA chimeras and their hybridization properties are described. The substitution of cytosine by 5-methylcytosine in DNA-PNA chimeras increased duplex stability while substitution of thymine by 5-bromouracil maintained it. Moreover, binding of DNA-PNA chimeras to double-stranded DNA to form triple helices was studied. In contrast to DNA, the presence of 5-methylcytosine and 5-bromouracil in DNA-PNA chimeras destabilized triple helix.     

Synthesis and properties of oligodeoxynucleotides containing 5-carboxy-2'-deoxycytidines

5-Carboxy-2'-deoxycytidine (dC(COO-)) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC(COO-)-G is less stable than that of the canonical C+-G pair and the Watson-Crick-type base pair of dC(COO-)-G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC(COO-) could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.     

Synthesis and its properties of oligonucleotide analogue containing thiocarbamate interlinkages.

Novel nucleotide analogues have been synthesized from morpholine subunits with thiocarbamate linkages. They indicated much stronger interaction with poly U or poly dT than the corresponding natural oligodeoxyribonucleotides. Solubility of the analogues in water was greatly enhanced by introducing sulfate groups at their both ends.     

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides

Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5′-amino-5′-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5′-amino-2′,5′-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5′-azido-2′,5′-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5′-amino-5′-deoxythymidine to the corresponding 5′-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension–cleavage procedure in genomic sequence analysis is discussed.     

Synthesis and structural study of quadruplex structures containing 2'-deoxy-8-methyladenosine

The synthesis and preliminary structural studies of ODNs A8MeGGGT and TA8MeGGGT, where A8Me represents 2'-deoxy-8-methyladenosine, are reported.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Synthesis of some oligodeoxynucleotides containing the C-nucleoside and 2'-deoxy-2'-fluoronucleoside moieties.

An automated computer-controlled, multipurpose synthesizer, featuring a novel method for the transport of liquids, was constructed and used in the synthesis of oligomers containing some C-nucleoside and 2'-deoxy-2'-fluoronucleoside moieties by the H-phosphonate procedure. The synthetic method and some prospects for biological use are outlined.     

Synthesis of 5'-deoxy-5'-nucleosideacetic acid derivatives

The synthesis of several new 5'-deoxy-5'-nucleosideacetic acid derivatives by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5'-aldehydes is described.     

Water mediated Dickerson-Drew-type crystal of DNA dodecamer containing 2'-deoxy-5-formyluridine

To investigate the role of divalent cations in crystal packing, a Dickerson-Drew-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2'-deoxy-5-formyluridine at X, was crystallized under several conditions with Ba(2+) ion instead of Mg(2+) ion. The crystal structure is isomorphous with the original Dickerson-type crystal containing Mg(2+) ion. In the Mg(2+)-free crystals, however, a five-membered ring of water molecules occupies the same position as the magnesium site found in the Mg(2+)-containing crystals, and connects the two duplexes similarly to the hydrated Mg(2+) ion. It has been concluded that the five-membered water molecules can take the place of the hydrated magnesium cation in crystallization. The 5-formyluracil residues form the canonical Watson-Crick pair with the opposite adenine residues.     

Synthesis and properties of ApU analogues containing 2'-halo-2'-deoxyadenosines. Effects of 2' substituents on oligonucleotide conformation

Five A-U analogues containing deoxyadenosine or 2'-halo-2'-deoxyadenosines, which are known to have widely different C3'-endo conformer populations according to their electronegativities of the halogen substituents, dAfl-U, dAcl-U, dAbr-U, dAio-U, and dA-U, were synthesized chemically. Characterization of these dimers has been performed by UV absorption, circular dichroism, and proton nuclear magnetic resonance spectroscopy. The results show that the dimers containing 2'-halo-2'-deoxyadenosines have stacked conformations with a geometry similar to that of A-U and the degree of stacking decreases in the order dAfl-U greater than dAcl-U greater than dAbr-U greater than dAio-U. dAcl-U is assumed to have the same degree of stacking as A-U. dA-U takes a more stacked conformation than does dAio-U, but the mode of stacking is different from those of the other dimers. The effects of the 2' substituents on dimer conformation are discussed in terms of electronegativity, molecular size, and hydrophobicity.