Convenient synthesis of oligodeoxynucleotides containing 2'-deoxy-6-thioinosine

A facile synthesis of oligodeoxynucleotides (ODN) containing 2'-deoxy-6-thioinosine (dI6S) based on the convertible nucleoside O6-phenyl-2'-deoxyinosine is presented. After standard solid-phase DNA synthesis and removal of the cyanoethyl protecting groups with DBU treatment with aqueous sodium hydrogen sulfide introduces the sulfur functionality, deprotects the other nucleobases and cleaves the ODN from the solid support in a one-pot reaction. In addition, the extinction coefficient of 2'-deoxy-6-thioinosine is determined by enzymatic fragmentation of the resulting ODN in the presence of adenosine deaminase.     

A convenient synthesis of 2'-deoxy-2-fluoroadenosine; a potential prodrug for suicide gene therapy

A convenient synthesis of 2'-deoxy-2-fluoro-adenosine (1) is described. Deaminative fluorination of 2-aminoadenosine (2) followed by silylation of the 3', 5'-hydroxyl groups gave the corresponding 2-fluoroadenosine derivative 4 in good yield. Thiocarbonylation of 4 to thiocarbonylimidazolyl derivative 5a followed by treatment with an excess of tris(trimethylsilyl)silane (TTMSS) and tert-butyl peroxide in toluene at 80 degrees C was found to affect an efficient deoxygenation to the corresponding 2'-deoxy derivative 6. Desilylation of 6 by Et4NF in CH3CN afforded 1 in high yield.     

The preparative method for 2-fluoroadenosine synthesis

The preparative method for the synthesis of 2-fluoroadenosine starting from commercially available guanosine was developed. It included the intermediate formation of 2-amino-6-azido-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine, which was isolated exclusively in the tetrazolo[5,1-i]-form {5-amino-7-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-7H -tetrazolo[5,1-i]purine}. The latter compound was converted by the Schiemann reaction to 6-azido-2-fluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine, which was isolated at an 80% yield after careful optimization of the process. The IR and ¹H NMR spectroscopy data indicated the 6-azido-2-fluoropurine structure of the aglycone. The catalytic reduction of the azido group in 6-azido-2-fluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine to the amino moiety and the subsequent deacetylation by the routine procedure resulted in 2-fluoroadenosine at a total yield of 74%.     

Scaleable and efficient synthesis of 2'-deoxy-2'-N-phthaloyl nucleoside phosphoramidites for oligonucleotide synthesis

2'-Deoxy-2'-N-phthaloyl nucleosides were prepared from arabino nucleosides by triflate displacement with phthalimide in the presence of DBU. The corresponding phosphoramidites suitable for automated oligonucleotide synthesis were also synthesized. The scalability of described procedures was demonstrated on a 100-g scale preparation of 2'-deoxy-2'-amino-C phosphoramidite.     

Convenient synthesis of 8-amino-2'-deoxyadenosine

We studied the behaviour of 8-azido-2'-deoxyadenosine and 8-bromo-2'-deoxyadenosine in aqueous solutions of ammonia and primary and secondary amines. Unexpectedly, 8-Azido-2'-deoxyadenosine is converted to 8-amino-2'-deoxyadenosine in excellent yields. The use of this reaction for the preparation of 8-aminoadenine derivatives needed for the preparation of oligonucleotides carrying 8-aminoadenine is discussed.     

Studies directed toward the synthesis of 2'-deoxy-2'-substituted arabino nucleosides

Synthesis of the C-nucleoside, 5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1-methyluracil, isosteric to the potent antiviral and anticancer nucleoside, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1-methyluracil (2'-fluoro-5-methyl-ara-U or FMAU) was achieved by exploitation of the 4,5'-anhydro-nucleoside. Attempts at application of this ribosyl-to-arabinosyl pyrimidine transformation to 2,5'-anhydrouridine resulted in the formation of 2,2'-anhydro-5-substituted-arabinosyluracil.     

Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.     

Chemical and enzymatic synthesis and antiviral properties of 2'-deoxy-2'-fluoroguanosine.

Chemical and enzymatic methods were employed for the synthesis of the title compound, 2'F-Guo 7. High antiviral activity of 2'F-Guo was established in chick embryo cells infected with influenza virus FPV/Rostock/34 (H7N1) and herpes simplex virus (HSV) type I (1C strain).     

Chemical stability of 2'-deoxy-5-methylisocytidine during oligodeoxynucleotide synthesis and deprotection

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-methylisocytidine 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.     

Convenient synthesis of sulfated oligofucosides

Two types of sulfated octyl tetra- to octaoligofucosides with different sulfation patterns were synthesized employing a combination of stepwise elongation and convergent strategies in which trichloroacetimidates and thioglycosides were selected as the glycosyl donors.     

Solid-phase synthesis of 5'-deoxy-5'-amino-clitocine analogues

Several 6-substituted-amino-5'-deoxy-5'-amino-clitocine analogues were synthesized in a parallel fashion in solid phase. The desired scaffold was generated by coupling 2,3-O-bis-(t-butyldimethylsilyl)-5-N-(monomethoxytrityl-polystyrene-resin)-1,5-diamino-5-deoxy-beta-D-ribofuranose and 4, 6-dichloro-5-nitropyrimidine. The scaffold was then reacted with a variety of amines to generate a small library of 14 analogues of 5'-deoxy-5'-amino-clitocine following a protocol developed earlier.     

Novel triazole 2'-deoxy-4'-thionucleosides: stereoselective synthesis and biological evaluation.

A study on the use of protecting groups led to the employment of the para-methoxybenzoyl (pMB) group as a directing group in the synthesis of novel triazole 2'-deoxy-4'-thionucleosides. Use of the pMB group gave alpha:beta ratios of 1:6 in the glycosylation step with azidotrimethylsilane. A series of novel triazoles were generated for in vitro antiviral evaluation.     

2-fluoro-ATP: a toxic metabolite of 9-beta-D-arabinosyl-2-fluoroadenine

Murine P388 cells incubated in vitro with the anticancer drug arabinosyl 2-fluoroadenine accumulate its 5'-triphosphate, F-araATP, as the major phosphorylated metabolite. A new chromatographically separate metabolite that accumulated to levels 10% of that of F-araATP was identified as 2-fluoro-ATP, by the following criteria. 1. The metabolite coeluted with the authentic compound on anion-exchange HPLC. 2. Dephosphorylation of the metabolite yielded a compound that was chromatographically identical to 2-fluoroadenosine. 3. The compound was sensitive to NaIO4 oxidation. Cellular incubation experiments indicated that 2-fluoroadenine, but not arabinosyl 2-fluorohypoxanthine, was the likely intermediate in the formation of 2-fluoro-ATP.     

Convenient synthesis of L-proline benzyl ester

Mesylates or tosylates of delta-hydroxy-L-norvaline esters spontaneously afford L-proline esters upon exposure to aqueous buffer in near quantitative yield. This novel reaction has led to the development of a simple route to optically active proline esters.     

Design, synthesis and biological evaluation of 2'-deoxy-2',2'-difluoro-5-halouridine phosphoramidate ProTides

We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.     

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.     

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.