Synthesis and NMR Assignment of the Two Diastereomers of 8,6′-Cyclo-2′,6′-Dideoxyadenosine

We herein present the first synthesis and characterization of the two C5′ diastereomers of 8,6′-cyclo-2′,6′-dideoxyadenosine. Starting from commercially available 2′-deoxyadenosine, the target cyclonucleosides were synthesized in 11 linear steps. Following a zinc-mediated cyclization reaction to form the seven-membered ring, the stereochemistry of the newly formed chiral center was established using two-dimensional NOESY NMR experiments.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids for the free supplemental resource.]     

Synthesis and Characterization of Oligodeoxynucleotides Containing 5′,8-Cyclopurine-2′-Deoxyribonucleosides

Abstract

Oligodeoxynucleotides containing the two 5′R and 5′S diastereoisomers of 5′,8-cyclo-2′-deoxyadenosine (CyclodAdo) and 5′,8-cyclo-2′-deoxyguanosine (CyclodGuo) have been synthesized using the phosphoramidite chemistry. The structural assignment and a few biochemical features of these modified DNA fragments are reported.     

Time And Concentration Dependence Of Fenton-Induced Oxidation Of dG

The influence of incubation time and Fenton reagent concentrations was investigated on the oxidation of 2′-deoxyguanosine. The compounds identified and quantified, through use of an LC-MS/MS system, were 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8,5′-cyclo-2′-deoxyguanosine (8,5′cyclodG) and the secondary oxidation products guanidinohydantoin and dehydro-guanidinohydantoin. 8-oxodG and 8,5′cyclodG formed very quickly, reaching a maximum rapidly, but with 8-oxodG a rapid decline occurred thereafter due to its further oxidation into the secondary products, which formed more slowly. Due to the better stability, 8,5′cyclodG correlated better with the general level of oxidation than 8-oxodG. The results emphasize the advantages of measuring other oxidation adducts than 8-oxodG alone.     

Nucleosides and Nucleotides. 232. Synthesis of 2′-C-Methyl-4′-thiocytidine: Unexpected Anomerization of the 2′-Keto-4′-thionucleoside Precursor

The synthesis of 2′-C-methyl-4′-thiocytidine (16) is described. Since the 2′-keto-4′-thiocytidine derivative 2β unexpectedly isomerized to 2α and the methylation of 2β proceeded predominantly from the less hindered α-face to give 7, the desired product 16 was synthesized via the Pummerer reaction of the sulfoxide 14 and N ⁴ -benzoylcytosine.     

Synthesis of 2′-O-Photocaged Ribonucleoside Phosphoramidites

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2?′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2′-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl < 4,5-dimethoxy-2-nitrobenzyl < 2′-O-o-nitrobenzyl.     

A New Route for the Synthesis of 4-Amino-5-Fluoro-7-(2′-Deoxy-2′-Fluoro-2′-C-Methyl-β-d- Ribofuranosyl)-1H-Pyrrolo[2,3-D]Pyrimidine

A new route for the synthesis of the anti-HCV nucleoside analogue, 4-amino-5-fluoro-7-(2′-deoxy-2′-fluoro-2′-C-methyl-β-d-ribofuranosyl)-1H-pyrrolo[2,3-d]pyrimidine 1, was developed.     

Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.     

Structure of 2′,5′-Linked Tetraribonucleotide Loops: A Novel RNA Motif

Abstract

We report on the three dimensional structure of an RNA hairpin containing a 2′,5′-linked tetraribonucleotide loop, namely, 5′-rGGAC(UUCG)GUCC-3′ (where UUCG = U_2′p5′U_2′p5′C_2′p5′G_2′p5′). We show that the 2′,5′-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3′,5′-linked RNA loop. The 2′,5′- RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar–base contacts, all of which contribute to the extra stability of this hairpin structure.     

Synthesis and Anti-HIV Activity of β-D-3′-Azido-2′,3′-unsaturated Nucleosides and β-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.     

Synthesis and Evaluation of 2′-Deoxy-2′-Spirodiflurocyclopropyl Nucleoside Analogs

The preparation of 2′-deoxy-2′-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2′-position. Then, a series of novel 2′-deoxy-2′-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.     

(5′R)-and (5′S)-purine 5′,8-cyclo-2′-deoxyribonucleosides: reality or artifactual measurements? A reply to Chatgilialoglu’s comments (this issue)

Abstract

This rebuttal letter is aimed at refuting the poor and false arguments elaborated by Chatgilialoglu (preceding article) in his response to the position article (Cadet et al. Free Radic Res 2019;53:574–577) that focussed on the putative reliability of the HPLC-MS/MS measurements of five radiation-induced damage to cellular DNA, which included 8-oxo-7,8-dihydro-2'-deoxyadenosine and the (5′R) and (5′S) diastereomers of 5′,8-cyclo-2′-deoxyadenosine and 5′,8-cyclo-2′-deoxyadenosine (Krokidis et al. Free Radic Res 2017;51:470–482). Unfortunately, none of the main issues we raised on the suitability of the analytical approach and the shortcomings associated with DNA extraction in HPLC based measurement methods of oxidatively generated damage in cells were properly considered in Chatigilialolu’s letter. The main questionable issues include the lack of information on the sensitivity of HPLC-MS/MS analysis, the absence of a dose curve that is essential in the formation of damage and the nonconsideration of artifactual oxidation.     

Study on Disulfur-Backboned Nucleic Acids: Part 3. Efficient Synthesis of 3′,5′-Dithio-2′-Deoxyuridine and Deoxycytidine

A general method is described for synthesizing 3′,5′-dithio-2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′-deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl-3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH^? ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 2-Pyridone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O,4′-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

A novel nucleoside hydrolase from Lactobacillus buchneri LBK78 catalyzing hydrolysis of 2′-O-methylribonucleosides

2′-O-Methylribonucleosides (2′-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2′-OMe-NRs, Lactobacillus buchneri LBK78 was found to decompose 2′-O-methyluridine (2′-OMe-UR). The enzyme responsible was partially purified from L. buchneri LBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2′-OMe-UR to 2′-O-methylribose and uracil, and the kinetic constants were K_m of 0.040 mM, k_cat of 0.49 s^?1, and k_cat/K_m of 12 mM^?1 s^?1. In a substrate specificity analysis, LbNH preferred ribonucleosides and 2′-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2′-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes.     

Effective Anomerisation of 2′‐Deoxyadenosine Derivatives During Disaccharide Nucleoside Synthesis

The formation of a disaccharide nucleoside (11) by O3′‐glycosylation of 5′‐O‐protected 2′‐deoxyadenosine or its N ⁶‐benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding α‐anomeric product (12). The latter reaction can be explained by instability of the N‐glycosidic bond of purine 2′‐deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2′‐deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3′‐O‐β‐d‐ribofuranosyl‐2′‐deoxyadenosines and its α‐anomer.     

Semisynthesis of 6-Chloropurine-2′-deoxyriboside 5′-Dimethoxytrityl 3′-(2-Cyanoethyl-N,N-diisopropylamino)Phosphoramidite and its Use in the Synthesis of Fluorescently Labeled Oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2′-deoxyriboside from the reaction of 6-chloropurine with 2′-deoxycytidine catalyzed by nucleoside-2′-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2′-deoxyriboside 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

Reaction of the 2′-Silyl and 2′-Stannyl Derivatives of 6-(Bromomethyl)Dimethylsilyl-1′,2′-Unsaturated Uridine Under Radical Conditions

The mode of cyclization (5-exo versus 6-endo) of 2-sila-5-hexen-1-yl radicals generated from 2′-tributylstannyl- and 2′-trimethylsilyl-6-(bromomethyl)dimethylsilyl-1′,2′-unsaturated uridines (8 and 9) was investigated. Although the actual structure of the reaction products differ from each other, reflecting the ease of elimination of the 2′-substituent, it was found that both substrates prefer the 5-exo-cyclization pathway.     

A New Odorless Procedure for the Synthesis of 2′-Deoxy-6-Thioguanosine and Its Incorporation into Oligodeoxynucleotides

6-S-[2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxy-6-thiogua nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl 3-mercapto-propionate. The phosphoramidite precursor derived from 2 was successfully applied to an automated DNA synthesizer to produce 2′-deoxy-6-thioguanosine containing ODN. The results showed that 2-ethylhexyl 3-mercaptopropionate is useful as an odor less reagent and also as an S-protecting group of 2′-deoxy-6-thioguanosine.     

7-Deaza-2′-Deoxyxanthosine: Nucleobase Protection and Base Pairing of Oligonucleotides

Oligonucleotides containing 7-deaza-2′-deoxyxanthosine (1) and 2′-deoxyxanthosine (2) were prepared. The 2-(4-nitrophenyl)ethyl group is applicable for 7-deazaxanthine protection that is removed with DBU by β-elimination, while the deprotection of the allyl residue with Pd (0) catalyst failed. Contrarily, the allyl group was found to be an excellent protecting group for 2′-deoxyxanthosine (2). The base pairing of nucleosides 1 and 2 with the four canonical DNA constituents as well as with 3 within the 12-mer duplexes is studied.     

Evaluation of 3-(4′-(4″-fluorophenyl)-6′-phenylpyrimidin-2′-yl)-2-phenylthiazolidin-4-one for in vivo modulation of biomarkers of chemoprevention in the 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

A new bis heterocycle comprising both bioactive 2-aminopyrimidine and thiazolidin-4-one nuclei namely 3-(4′-(4″-fluorophenyl)-6′-phenylpyrimidin-2′-yl)-2-phenylthiazolidin-4-one 3 was synthesized, characterized with the help of melting point, elemental analysis, FT-IR, MS, one-dimensional NMR (¹H, ¹³C) spectra and we evaluated the chemopreventive potential of 3-(4′-(4″-fluorophenyl)-6′-phenylpyrimidin-2′-yl)-2-phenylthiazolidin-4-one based on in vivo inhibitory effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Administration of 3 effectively suppressed oral carcinogenesis initiated with DMBA as revealed by the reduced incidence of neoplasms. Lipid peroxidation, glutathione (GSH) content, and the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) were used to biomonitor the chemopreventive potential of 3. Lipid peroxidation was found to be significantly decreased, whereas GSH, GPx, GST, and GGT were elevated in the oral mucosa of tumor-bearing animals. Our data suggest that 3 may exert its chemopreventive effects in the oral mucosa by modulation of lipid peroxidation and enhancing the levels of GSH, GPx, and GST.