SYNTHESIS AND PROPERTIES OF OLIGONUCLEOTIDES CONTAINING 8-BROMO-2′-DEOXYGUANOSINE

The preparation of oligonucleotides containing 8-bromo-2′-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2′-deoxyguanosine paired with natural bases were much less stable.     

2′-MODIFIED OLIGONUCLEOTIDES FROM METHOXYOXALAMIDO AND SUCCINIMIDO PRECURSORS: SYNTHESIS,PROPERTIES, AND APPLICATIONS

Synthesis of 2′-modified oligonucleotides from 2′-methoxyoxalamido (MOX) and 2′-succinimido (SUC) precursors is described. Their physical and biochemical properties were assessed. Synthesized oligonucleotides were used as primers in advanced DNA sequencing protocols. An example of sequencing directly off genomic DNA template without prior cloning or PCR amplification is presented.     

SYNTHESIS OF 3′-DEOXY-3′-C-HYDROXYMETHYL ANALOGUES OF TIAZOFURIN AND RIBAVIRIN

On the basis of potent biological activity of 3′-branched-3′-deoxynucleoside analogues, novel ribavirin and tiazofurin derivatives with 3′-C-hydroxymethyl substituent were synthesized, starting from D-xylose.     

CHEMICAL STABILITY OF 2′-DEOXY-5-METHYLISOCYTIDINE DURING OLIGODEOXYNUCLEOTIDE SYNTHESIS AND DEPROTECTION

ABSTRACT

Previous studies have encountered difficulties with degradation of some isocytidine derivatives during solid-phase synthesis and deprotection of oligonucleotides. Here we investigate the degradation of a commonly used derivative, 2′-deoxy-5-methylisocytidine, during oligodeoxynucleotide synthesis and deprotection. A small, but detectable amount of hydrolytic deamination occurred at ca. 0.5% of 2′-deoxy-5-methyliso-cytidine residues using routine synthesis and deprotection conditions. Depyrimidination, or cleavage of the glycosylic bond, occurred to a far lesser extent during alkaline deprotection than previously suggested. In contrast to model studies of nucleoside monomers, significant depyrimidination was not observed, even at extended incubation times.     

SYNTHESIS AND STRUCTURAL STUDY OF QUADRUPLEX STRUCTURES CONTAINING 2′-DEOXY-8-METHYLADENOSINE

The synthesis and preliminary structural studies of ODNs A^8MeGGGT and TA^8MeGGGT, where A^8Me represents 2′-deoxy-8-methyladenosine, are reported.     

ANTISENSE PROPERTIES OF 2′-O-DIMETHYLAMINOOXYETHYL (2′-O-DMAOE) OLIGONUCLEOTIDES

Antisense oligonucleotides with 2′-O-{2-[N,N-dimethyl)aminooxy]ethyl} or (2′-O-DMAOE) modification were synthesized and evaluated for nuclease resistance and pharmacology both in vitro and in vivo. This modification exhibits very high nuclease resistance and efficacy in various biological (ICAM-1, C-raf and PKC-α) targets.     

SYNTHESIS OF SOME 2′-FLUORO-2′-DEOXY-3′-C-ETHYNYL AND 3′-C-VINYL-β-D-LYXOFURANOSYL NUCLEOSIDES

1-(2-Fluoro-2-deoxy-β-D-arabinofuranosyl)uracil (5) and 1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)cytosine (6) were synthesized as reported earlier. Both of these compounds were converted into 2′-fluoro-2′-deoxy-3′-C-ethynyl and 3′-C-vinyl-β-D-lyxofuranosyl nucleosides (16–19) by a multistep sequence. All these new nucleosides were evaluated against seven human tumor cell lines in vitro.     

SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D- and L-2′-DEOXY-2′-FLUORORIBONUCLEOSIDES

Based on the discovery of β-D-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of β-D- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2′-fluoro group was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely β-D-2′-deoxy-2′,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As β-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2′-fluoro were combined into one molecule, yielding β-D-2′-deoxy-2′-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2′-DEOXY-4′-THIO-PYRAZOLO[3,4-d]PYRIMIDINE NUCLEOSIDES

The coupling of 4-aminopyrazolo [3, 4-d]pyrimidine with the appropriate thio sugar gave a 3:1 ratio of α,β blocked 4-amino-1-(2-deoxy-4-thio-D-erythropentofuranosyl)- 1H pyrazolo[3,4-d]pyrimidine nucleosides. The mixture was deblocked, both the anomers were separated, and the β-anomer was readily deaminated by adenosine deaminase. The nucleosides have been characterized, and their anomeric configurations have been determined by proton NMR. All three nucleosides were evaluated against a panel of human tumor cell lines for cytotoxicity in vitro. The details of a convenient and high yielding synthesis of these nucleosides are described.     

SYNTHESIS AND STUDY OF CONFORMATIONALLY RESTRICTED 3′-DEOXY-3′,4′-EXO-METHYLENE NUCLEOSIDE ANALOGUES

Hitherto unknown restricted 3′-deoxy-3′,4′-exo-methylene nucleoside derivatives bearing the nucleic acid naturally occurring pyrimidine bases have been synthesized. The compounds were tested for their activity against HIV, HBV, and several RNA viruses, but they did not show significant antiviral effect.     

THE SYNTHESIS OF,AND STUDIES ON, 2′-C-MODIFIED NUCLEOTIDES

The synthesis of uridine monomers containing either a 2′-deoxy-2′-C-methy- lcyano or ethylcyano group is described. These monomers are intended for incorporation into oligonucleotides to investigate a proposed duplex-stabilising effect exerted by 2′-tethered amide groups.     

MECHANISM OF INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE BY 5′-DEOXY-5′-S-ALLENYLTHIOADENOSINE AND 5′-DEOXY-5′-S-PROPYNYLTHIOADENOSINE

5′-Deoxy-5′-S-allenylthioadenosine 1 and 5′-deoxy-5′-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II “mechanism-based” inhibitors.     

INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE WITH FLUORINATED ANALOGS OF 2′- AND 3′-DEOXY-5′-METHYLTHIOADENOSINE

Fluorinated analogs of 2′- and 3′-deoxy-5′-methylthioadenosine 1–4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

SYNTHESIS OF OLIGONUCLEOTIDES BEARING AN ARYLAMINE MODIFICATION IN THE C8-POSITION OF 2′-DEOXYGUANOSINE

C8-Arylamine-dG adducts were converted into their corresponding 5′-O-DMTr-3′-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. The oligonucleotides were studied for their CD properties, T_m values, and their effects on primer extension assays using human DNA-polymerase β.     

SYNTHESIS OF CARBOCYCLIC ANALOGS OF 2′,3′-DIDEOXYSANGIVAMYCIN, 2′,3′-DIDEOXYTOYOCAMYCIN,AND 2′,3′-DIDEOXYTRICIRIBINE

Syntheses and antiviral activity of new carbocyclic analogs of 2′, 3′-dideoxysangivamycin, 2′,3′-dideoxytoyocamycin and 2′,3′-dideoxytriciribine is described. The key intermediate, carbocyclic 4-chloro- 5-iodopyrrolopyrimidine, was synthesized in good yield via a novel iodination method using I₂ and CF₃COOAg. This carbocyclic 4-chloro-5-iodopyrrolopyrimidine then allowed for a concise synthesis of the desired 4,5-disubstituted carbocyclic nucleosides.     

SYNTHESIS AND PROPERTIES OF A NOVEL BRIDGED NUCLEIC ACID ANALOGUE, 5′-AMINO-3′,5′-BNA

An oligonucleotide P3′?N5′ phosphoramidate (5′-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5′-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3′?N5′ phosphoramidate linkage and a constrained sugar moiety, 5′-amino-3′-C,5′-N-methylene bridged nucleic acid (5′-amino-3′,5′-BNA). The binding affinity of the 5′-amino-3′,5′-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5′-amino-3′,5′-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5′-amino-DNA.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4′-C-HYDROXYMETHYL-2′-FLUORO-D-ARABINOFURANOSYLPURINE NUCLEOSIDES

A series of 4′-C-hydroxymethyl-2′-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity in human tumor cell lines. A convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-α-D-arabinofuranosyl bromide (13) was developed. Coupling of 13 with the sodium salt of 2,6-dichloropurine led to five target purine nucleosides.     

2′-C-ALKOXY AND 2′-C-ARYLOXY DERIVATIVES OF N-(2-PHOSPHONOMETHOXYETHYL)-PURINES AND -PYRIMIDINES: SYNTHESIS AND BIOLOGICAL ACTIVITY

A series of novel, unusual type of acyclic phosphonate-based nucleotide analogues related to well-known antivirals (PMEA and HPMPA) was synthesized using easily available synthon. These compounds, which are distinguished for the presence of phosphonomethyl acetal linkage, form a group of derivatives that contribute to the understanding of structure-activity relationship within the area of acyclic nucleotide analogues.     

SYNTHESIS OF 6-FORMYLURIDINE 5′-MONOPHOSPHATE: 6-FORMYL-UMP

A synthesis of 6-formyluridine 5′-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5′-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.