Investigation of reactions postulated to occur during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Model 3'-azido-3'-deoxynucleosides with thiol or vicinal dithiol substituents at C2' or C5' were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides. Esterification of 5'-(tert-butyldiphenylsilyl)-3'-azido-3'-deoxyadenosine and 3'-azido-3'-deoxythymidine (AZT) with 2,3-S-isopropylidene-2,3-dimercaptopropanoic acid or N-Boc-S-trityl-L-cysteine and deprotection gave 3'-azido-3'-deoxy-2'-O-(2,3-dimercaptopropanoyl or cysteinyl)adenosine and the 3'-azido-3'-deoxy-5'-O-(2,3-dimercaptopropanoyl or cysteinyl)thymidine analogs. Density functional calculations predicted that intramolecular reactions between generated thiyl radicals and an azido group on such model compounds would be exothermic by 33.6-41.2 kcal/mol and have low energy barriers of 10.4-13.5 kcal/mol. Reduction of the azido group occurred to give 3'-amino-3'-deoxythymidine, which was postulated to occur with thiyl radicals generated by treatment of 3'-azido-3'-deoxy-5'-O-(2,3-dimercaptopropanoyl)thymidine with 2,2'-azobis-(2-methyl-2-propionamidine) dihydrochloride. Gamma radiolysis of N(2)O-saturated aqueous solutions of AZT and cysteine produced 3'-amino-3'-deoxythymidine and thymine most likely by both radical and ionic processes.     

Stereospecific syntheses of 3'-deuterated pyrimidine nucleosides and their site-specific incorporation into DNA

[reaction: see text] 2'-Deoxy-3'-deutero pyrimidines have been synthesized in high yields and incorporated into deoxyoligonucleotides using standard phosphoramidite chemistry. A key synthetic step is a stereospecific reduction of 3'-keto nucleosides using sodium triacetoxyborodeuteride to give 3'-deuterated thymidine and 2'-deoxy uridine nucleosides. Conversion of the corresponding phorphoramidites 7a and 7b to 4-triazolo derivatives has, for the first time, enabled incorporation of 2'-deoxy-3'-deutero cytidine and 2'-deoxy-3'-deutero-5-methyl cytidine into oligonucleotides.     

Deoxy Derivatives of L-like 5'-Noraristeromycin

Several base variations of 2'- and 3'-deoxy derivatives of (+)-4'-deoxy-5'-noraristeromycin have been prepared from enantiomerically pure precursors following standard purine nucleoside construction. These carbocyclic nucleosides were evaluated against hepatitis B virus (HBV) and found to be inactive. No cytotoxicity to the cell line was observed.     

Synthesis and hybridization properties of 2'-O-methylated oligoribonucleotides incorporating 2'-O-naphthyluridines

2'-O-(1-Naphthyl)uridine and 2'-O-(2-naphthyl)uridine were synthesized by a microwave-mediated reaction of 2,2'-anhydrouridine with naphthols. Using the 3'-phosphoramidite building blocks, these 2'-O-aryluridine derivatives were incorporated into 2'-O-methylated oligoribonucleotides. Incorporation of five 2'-O-(2-naphthyl)uridines into a 2'-O-methylated RNA sense strand significantly increased the thermostability of the duplex with a 2'-O-methylated RNA antisense strand. Circular dichroism spectroscopy and molecular dynamic simulation of the duplexes formed between the modified RNAs and 2'-O-methyl RNAs suggested that there are π-π interactions between two neighboring naphthyl groups in a sequence of the five consecutively modified nucleosides.     

Synthesis, spectroscopic characterization, axial base coordination equilibrium and photolytic kinetics studies of a new coenzyme B12 analogue-3'-deoxy-2',3'-anhydrothymidylcobalamin

A new coenzyme B12 (AdoCbl) analogue, 3'-deoxy-2',3'-didehydrothymidylcobalamin (2',3'-anThyCbl) was prepared by the reaction of 5'-iodo-3'-deoxy-2',3'-dihydrothmidine with reduced B12a, and characterized by UV-Vis, CD, ESI-MS and NMR spectroscopies. Its axial base (dbzm) coordination equilibria with pH's and temperatures were investigated and showed similar features to those of coenzyme B12. Photolytic dynamics studies under homolytic and heterolytic conditions demonstrated that the Co-C bond of the analogue is slightly more photolabile relative to coenzyme B12.     

Negative-ion mass spectrometry of substituted adenine bases and adenosine nucleosides

The negative-ion chemical ionization (ammonia, 5 Pa source pressure) mass spectra of a series of substituted adenine bases, adenosine nucleosides, and the trimethylsilyl derivatives of the nucleosides are described. Selected ions from these spectra were subject to collisionally activated dissociation with mass-analysed ion kinetic energy (CAD/MIKE) analysis of the products and the spectra assessed for information content. In addition to observing strong peaks due to quasimolecular ions and heterocyclic-base ions, it proved possible to differentiate between 2'-, 3'- and 5'-deoxy and between 2'- and 3'-O-methyl isomers. The negative-ion chemical ionization spectra of four methyladenines are essentially identical, but could be clearly distinguished from each other by CAD/MIKE analysis.     

Nucleosides XI. Synthesis and antiviral evaluation of 5'-alkylthio-5'-deoxy quinazolinone nucleoside derivatives as S-adenosyl-L-homocysteine analogs

4-amino-1-(beta-D-ribofuranosyl)quinazolin-2-one (3) was prepared by a direct glycosylation of 4-aminoquinazolin-2-one (7) using the Vorbruggen's silylation method and provided exclusively the beta-anomer. This quinazoline nucleoside and its 2',3'-O-isopropylidene derivative (9) did not undergo the coupling reaction with dialkyl disulfides in the presence of tri-n-butylphosphine unless their 4-amino groups were protected by N,N-dimethylaminomethylidene. This approach provides a viable alternative synthetic route to 5'-alkylthio-5'-deoxy nucleosides.     

Practical synthesis of 2'-deoxy-4'-thioribonucleosides: substrates for the synthesis of 4'-thioDNA

We report herein a practical synthesis of 4'-thiothymidine (15) and appropriately protected 2'-deoxy-4'-thiocytidine (16), -thioadenosine (27), and -thioguanosine (29) derivatives, substrates for the synthesis of 4'-thioDNA, from the corresponding 4'-thioribonucleosides. 2'-deoxy-4'-thiopyrimidine nucleosides were synthesized using a radical reaction of the corresponding 2'-alpha-bromo derivatives, which were prepared via 2,2'-O-anhydro derivatives. 2'-deoxy-4'-thiopurine nucleosides were synthesized using the same radical reaction of the corresponding 2'-beta-bromo derivatives.     

Syntheses of puromycin from adenosine and 7-deazapuromycin from tubercidin, and biological comparisons of the 7-aza/deaza pair.

Protection (O5') of 2',3'-anhydroadenosine with tert-butyldiphenylsilyl chloride and epoxide opening with dimethylboron bromide gave the 3'-bromo-3'-deoxy xylo isomer which was treated with benzylisocyanate to give the 2'-O-(N-benzylcarbamoyl) derivative. Ring closure gave the oxazolidinone, and successive deprotection concluded an efficient route to 3'-amino-3'-deoxyadenosine. Analogous treatment of the antibiotic tubercidin [7-deazaadenosine; 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine] gave 3'-amino-3'-deoxytubercidin. Trifluoroacetylation of the 3'-amino function, elaboration of the heterocyclic amino group into a (1,2,4-triazol-4-yl) ring with N,N'-bis[(dimethylamino)methylene]hydrazine, and nucleophilic aromatic substitution with dimethylamine gave puromycin aminonucleoside [9-(3-amino-3-deoxy-beta-D-ribofuranosyl)-6-(dimethylamino)purine] and its 7-deaza analogue. Aminoacylation [BOC-(4-methoxy-L-phenylalanine)] and deprotection gave puromycin and 7-deazapuromycin. Most reactions gave high yields at or below ambient temperature. Equivalent inhibition of protein biosynthesis in a rabbit reticulocyte system and parallel growth inhibition of several bacteria were observed with the 7-aza/deaza pair. Replacement of N7 in the purine ring of puromycin by "CH" has no apparent effect on biological activity.     

Synthesis of pyrimidine 2'-deoxy ribonucleosides branched at the 2'-position via radical atom-transfer cyclization reaction with a vinylsilyl group as a radical-acceptor tether

Recently, we developed a regio- and stereoselective method for introducing a vinyl group at the position beta to a hydroxyl group in halohydrins or alpha-phenylselenoalkanols via a radical atom-transfer cyclization reaction with a vinylsilyl group as a temporary connecting radical-acceptor tether. The synthesis of 2'-deoxy-2'-C-vinyl- and 2'-deoxy-2'-C-hydroxymethyluridines (7 and 8, respectively) and the corresponding 2'-deoxycytidine congeners (10 and 11, respectively), which were designed as potential antitumor and/or antiviral agents, was achieved using this radical atom-transfer cyclization as the key step. When the 2'-deoxy-2'-iodo-5'-O-monomethoxytrityl (MMTr) uridine derivative 19a, bearing a vinylsilyl group at the 3'-hydroxyl group, was heated with (Me(3)Sn)(2) and AIBN in benzene, the corresponding radical atom-transfer product was generated, which in turn was successively treated with tetrabutylammonium fluoride and TBSCl/imidazole to give the desired 2'-deoxy-5'-O-MMTr-3'-O-TBS-2'-C-vinyluridine (25). Compound 25 was successfully converted into the target 2'-deoxy-2'-branched pyrimidine ribonucleosides 7, 8, 10, and 11.     

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.     

Selectivity and affinity of DNA triplex forming oligonucleotides containing the nucleoside analogues 2'-O-methyl-5-(3-amino-1-propynyl)uridine and 2'-O-methyl-5-propynyluridine

Triplex forming oligonucleotides (TFOs) containing the nucleoside analogues 2'-O-methyl-5-propynyluridine (1) and 2'-O-methyl-5-(3-amino-1-propynyl)uridine (2) were synthesized. The affinity and selectivity of triplex formation by these TFOs were studied by gel shift analysis, T(m) value measurement, and association rate assays. The results show that the introduction of 1 and 2 into TFOs can improve the stability of the triplexes under physiological conditions. Optimized distribution of 1 or 2 in the TFOs combined with a cluster of contiguous nucleosides with 2'-aminoethoxy sugars resulted in formation of triplexes with further enhanced stability and improved selectivity.     

Nucleosides and nucleotides. 186. Synthesis and biological activities of pyrimidine carbocyclic nucleosides with a hydroxyamino group instead of a hydroxymethyl group at the 4'-position of the sugar moiety.

Pyrimidine carbocyclic nucleosides with a hydroxyamino group instead of a hydroxymethyl group at the 4'-position of the sugar moiety were designed as potential antitumor and/or antiviral agents. Pd (O)-catalyzed reactions of enantiomerically pure (+)-(1R,4S)-4-[(tert-butyldiphenylsilyl)oxy]-1-(ethoxycarbonylo xy)-2- cyclopentene (9) with N3-benzoylthymine and -uracil gave carbocyclic nucleosides 10 and 11. Subsequent Pd (O)-catalyzed reactions of N3-benzoyl-1-[(1R,4S)-4-(ethoxycarbonyloxy)-2-cyclopenten-1- yl]thymine (14) and -uracil (15) with O-benzylhydroxylamine smoothly gave the hydroxyamino-substituted carbocyclic nucleosides 16 and 17. From these nucleosides, the target compounds were prepared after deprotection or further reactions. The 2',3'-didehydro-2',3'-dideoxythymidine (D4T) analogue 20 was the most effective compound, with IC50 values of 27.3 and 34.5 microM against KB and L1210 cells in vitro. Carbocyclic analogues of uridine and cytidine (29 and 32) were less effective than 20 against both cell lines.     

Glucose-derived 3'-(carboxymethyl)-3'-deoxyribonucleosides and 2', 3'-lactones as synthetic precursors for amide-linked oligonucleotide analogues

Treatment of a 1,2-O-isopropylidene-3-ketopentofuranose derivative (obtained from D-glucose) with [(ethoxycarbonyl)methylene]triphenylphosphorane and catalytic hydrogenation of the resulting alkene gave stereodefined access to 3-(carboxymethyl)-3-deoxy-D-ribofuranose derivatives. Esters of 5-O-acetyl- or 5-azido-5-deoxy-3-(carboxymethyl)-D-ribofuranose were coupled with nucleobases to give branched-chain nucleoside derivatives. Ester saponification and protecting group manipulation provided 2'-O-(tert-butyldimethylsilyl) ethers of 5'-azido-5'-deoxy- or 5'-O-(dimethoxytrityl) derivatives of 3'-(carboxymethyl)-3'-deoxyribonucleosides that are effective precursors for synthesis of amide-linked oligoribonucleosides.     

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages

[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.     

从尿核苷合成异胞苷衍生物的简便有效方法

本文报道从5'-O-磺酰基-2',3'-O-异亚丙基尿核苷通过一步法合成N-烷基2',3'-O-异亚丙基异胞苷和从5'-O-对甲苯磺酰基尿核苷合成5'-脱氧-5'-异丙氨基尿核苷。并对以上产物形成的机制作了探讨。     

Synthesis of 5'-Methylene-Phosphonate Furanonucleoside Prodrugs: Application to D-2'-Deoxy-2'-α-fluoro-2'-β-C-methyl Nucleosides

A new and facile synthetic pathway to metabolically stable 5'-methylene-bis(pivaloyloxymethyl)(POM)phosphonate furanonucleoside prodrugs is reported. The key step involves a Horner-Wadsworth-Emmons reaction of a tetra(pivaloyloxymethyl) bisphosphonate salt with appropriately protected 5'-aldehydic nucleosides. This efficient approach was applied for the synthesis HCV related 2'-deoxy-2'-α-fluoro-2'-β-C-methyl nucleosides.     

2'-modified-2-thiothymidine oligonucleotides

[reaction: see text] Oligonucleotides with novel modifications, 2'-O-[2-(methoxy)ethyl]-2-thiothymidine and 2'-deoxy-2'-fluoro-2-thiothymidine, have been synthesized. These modified oligonucleotides exhibited very high thermal stability when hybridized with complementary RNA. 2-O-(2-Methoxy)ethyl-2-thiothymidine modified oligonucleotide phosphodiesters showed enhanced resistance toward nucleases (t(1/2) > 24 h), but 2'-deoxy-2'-fluoro-2-thiothymidine modified oligonucleotide phosphodiesters showed limited stability to nucleotytic degradation.     

O6-(benzotriazol-1-yl)inosine derivatives: easily synthesized, reactive nucleosides

A novel class of O6-(benzotriazol-1-yl)inosine as well as the corresponding 2'-deoxy derivatives can be conveniently prepared by a reaction between sugar-protected or -unprotected inosine or 2'-deoxyinosine nucleosides and 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The reaction appears to proceed via a nucleoside phosphonium salt, and in the absence of any additional nucleophile, the released 1-hydroxybenzotriazole undergoes reaction with the formed phosphonium salt leading to the requisite O6-(benzotriazol-1-yl)inosine or 2'-deoxyinosine derivatives. Isolation and characterization of the phosphonium salt as well as analysis by 31P{1H} NMR appear to be consistent with this reaction pathway. The resulting O6-(benzotriazol-1-yl)inosine derivatives are effective as electrophilic nucleosides, undergoing facile reactions with a variety of nucleophiles such as alcohols, phenols, amines, and a thiol. Unusual and challenging nucleoside derivatives such as an aryl-bridged dimer, a nucleoside-amino acid conjugate, and a nucleoside-nucleoside dimer have also been synthesized from the O6-(benzotriazol-1-yl)-2'-deoxyinosine derivative. Finally, a fully protected DNA building block, the O6-(benzotriazol-1-yl)-2'-deoxyinosine 5'-O-DMT 3'-O-phosphoramidite, has been prepared and a preliminary evaluation of its use for DNA modification has been performed. Results from these studies indicate several important facts: A single, simple methodological approach provides a class of stable, isolable ribo and 2'-deoxyribonucleoside derivatives that possess excellent reactivity for SNAr chemistry with a wide range of nucleophiles. Also, a benzotriazolyl nucleoside phosphoramidite appears to be a suitable reagent for incorporation into DNA for purposes of site-specific DNA modification.     

Direct glycosylation: synthesis of alpha-indoline ribonucleosides

A selective synthesis of α-anomers of indoline nucleosides is described. Ribonucleosides of indoline, dimethylindoline and 5-bromoindoline are readily prepared in good yield by reacting indoline bases directly with the protected sugar, 2,3-O-(1-methylethylidene) 5-O-(triphenylmethyl)-D-ribofuranose in dry ethanol or methylene chloride in presence of molecular sieves at 40-60 °C.