3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

New 3′-3′ Linkers for Alternate Strand Triplex Forming Oligonucleotides

Abstract

New solid supports, functionalized with suitably protected 1,2,3-propanetriol and cis,cis-1,3,5-cyclohexanetriol, were efficiently prepared and used in the standard automated synthesis of 3′-3′ linked ODNs for triplex formation experiments.     

A Short Syhthesis of 3′-(Methylsulfinyl)-3′-Deoxythymidine and Related Analogues

Abstract

The ring opening of the O-2,3′-anhydrothymidine 5 with the anion of methyl mercaptan gave the 3′-methylthio derivaative 6. Subsequent oxidation and deprotection afforded 3′-(methyl-sulfinyl)-3′-deoxythymidine 2 and its sulfone analogue 3.     

多核苷酸的3′-端磷酰化

本文报导了一种使寡聚核苷酸3'-端磷酰化的新方法。其原理是用多核苷酸磷酸化酶(PNPase),使7-甲基鸟苷5'二磷酸(m~7GDP)在引物存在下聚合,然后在温和的化学条件下,选择性消去7-甲基鸟苷(m~7G)。我们应用两种三核苷二磷酸CpCpA,CpUpC作引物,结果成功地合成了两种三核苷酸CpCpAp,CpUpCp。此方法还可用于多核苷酸的3'-~(32)P标记。     

多核苷酸的3′-端磷酰化

本文报导了一种使寡聚核苷酸3′-端磷酰化的新方法。其原理是用多核苷酸磷酸化酶(PNPase),使7-甲基鸟苷5′二磷酸(m~7GDP)在引物存在下聚合,然后在温和的化学条件下,选择性消去7-甲基鸟苷(m~7G)。我们应用两种三核苷二磷酸CpCpA,CpUpC 作引物,结果成功地合成了两种三核苷酸CpCpAp,CpUpCp。此方法还可用于多核苷酸的3′-~(32)P 标记。     

Synthesis of 3′-N-Substituted 3′-Amino-3′-Deoxythymidine Derivatives

Abstract

A series of 3′-N-substituted 3′-amino-3′-deoxythymidine derivatives with alkyl, alkenyl and alkylaryl substituents was synthesized by two methods. The first method involved the reaction of 1-(2,3-dideoxy-3-0-mesyl-5-0-trityl-β-D-threo-pentofuranosyl)thymine with an appropriate amine. In the second method, 3′-amino-5′-0-trityl-3′-deoxy-thymidine served as a synthetic precursor which was reacted with an appropiate aldehyde or ketone followed by sodium borohydride reduction. An improved synthesis of 3′-amino-3′-deoxythymidine from 3′ -azido-5′-0-trityl-3′-deoxythymidine using sodium borohydride was also described.     

Stereocontrolled Facile Synthesis and Biological Evaluation of (3′S) and (3′R)-3′-Amino (and Azido)-3′-Deoxy Pyranonucleosides

This article describes the synthesis of (3 ′S) and (3 ′R)-3 ′-amino-3 ′-deoxy pyranonucleosides and their precursors (3 ′S) and (3 ′R)-3 ′-azido-3 ′-deoxy pyranonucleosides. Azidation of 1,2:5,6-di-O-isopropylidene-3-O-toluenesulfonyl-α-D-allofuranose followed by hydrolysis and subsequent acetylation afforded 3-azido-3-deoxy-1,2,4,6-tetra-O-acetyl-D-glucopyranose, which upon coupling with the proper silylated bases, deacetylation, and catalytic hydrogenation, obtained the target 3 ′-amino-3 ′-deoxy-β-D-glucopyranonucleosides. The desired 1-(3 ′-amino-3 ′-deoxy-β-D-allopyranosyl)5-fluorouracil was readily prepared from the suitable imidazylate sugar after azidation followed by a protection/deprotection sequence and reduction of the unprotected azido precursor. No antiviral activity was observed for the novel nucleosides. Moderate cytostatic activity was recorded for the 5-fluorouracil derivatives.     

Chemoenzymatic Synthesis of 3′-Deoxy-3′-(4-Substituted-Triazol-1-YL)-5-Methyluridine

An efficient protocol has been developed for the synthesis of a small library of 3′-deoxy-3′-(4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction of 3′-azido-3′-deoxy-5-methyluridine with different alkynes under optimized condition in an overall yields of 76%–92%. Here, the azido precursor compound, i.e., 3′-azido-3′-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield. Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxycoumarins or naphthols with propargyl bromide in acetone using K₂CO₃in excellent yields. All synthesized compounds were unambiguously identified on the basis of their spectral (IR, ¹H-, ¹³C NMR spectra, and high-resolution mass spectra) data analysis.     

A Facile Synthetic Method for 3′-α-Fluoro- 2′,3′-dideoxyadenosine

Abstract

A facile method for the synthesis of 3′-α-fluoro-2′,3′-dideoxyadenosine (5) has been developed using a novel rearrangement of 3′-β-bromine to the 2′-β position during 3′-α fluorination.     

Synthesis of 3′-Amino-3′-deoxyguanosine 5′-Triphosphate

Abstract

Phosphorylation of 2′-0-acetyl-3′-trifluoroacetamido-3′-deoxy-N²-palmitoylguanosine with N-morpholino-O, O-bis(1-benzotriazolyl)phos-phate gives a 5′-phosphotriester. Removal of the benzotriazolyl group and addition of pyrophosphoric acid gave, after deblocking all protecting groups, GTP(3′NH₂).     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

紫色马铃薯类黄酮3，′5′-羟化酶基因（StF3′5′H）的克隆及分析

类黄酮3′,5′羟-化酶（ flavonoid 3′,5′-hydroxylase, F3′5′H）是植物花青素生物合成途径中的一个关键酶,紫色土豆（ Solanum tueb or sum） F3′5′H基因的克隆将为花青素合成调控和花青素代谢工程研究提供优质基因资源。研究采用RACE技术克隆了紫色土豆F3′5′H基因的cDNA全长序列,用生物信息学方法对其核苷酸和蛋白质序列进行了分析,并用半定量PCR 技术分析了F3′5′H基因在不同组织中的表达情况,同时研究了赤霉素和蔗糖处理后F3′5′H基因表达与花青素积累之间的相关性。研究结果表明,克隆的紫色土豆F3′5′H的cDNA全长为1854 bp,包含一个1530 bp的完整ORF,共编码509个氨基酸。生物信息学分析表明,StF3′5′H基因推测编码的氨基酸序列与其它植物的F3′5′H蛋白的相似性很高。 StF3′5′H基因的表达具有组织特异性,在紫色土豆根、茎和叶柄中都有表达,其中在叶柄中表达最强,而在块茎、叶轴和叶片中几乎检测不到StF3′5′H基因的表达。赤霉素和蔗糖能促进紫色土豆StF3′5′H基因的表达,进而促进花青素的积累。     

Oligomerizations of deoxyadenosine bis-phosphates and of their 3′-5′, 3′-3′, and 5′-5′ dimers: Effects of a pyrophosphate-linked,poly(t) analog

A pyrophosphate-linked polynucleotide analog based on thymidine 3,5 bis-phosphate (pTp) catalyzes the oligomerization of activated dimers of pdAp in the presence of MgCl₂. Although no catalysis of the oligomerization of the activated monomer (ImpdAplm) was observed in the presence of MgCl₂, there was a significant stimulation of oligomerization by the template in the presence of MnCl₂.     

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract

Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n²A) and uridine (U) residues, (2′-5′) n²ApU (1), (3′-5′) n²ApU (2), (2′-5′) Upn²A (3) and (3′-5′) Upn²A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n²ApU isomers have more stacked structure than the Upn²A isomers.     

Novel Spironucleosides: 1″,3″-Thiazolidine-2″-Spiro-3′-3′-Deoxyuridine Derivatives

Abstract

Reaction of 2′,5′-di-O-TBDMS-3′-ketouridine 1 with L-cysteine yielded in good yield a resolvable mixture of the two expected epimeric spironucleosides 2 and 3. Amidification of their carboxylic group took place readily and the ribo carboxamide 4 was oxidized to the corresponding sulfoxide 6. Despite their similarity to TSAO derivatives these compounds did not exhibit usable anti-HIV activity.     

鹤望兰类黄酮3′,5′-羟化酶基因SrF3′5′H的克隆及表达分析

采用RT-PCR和RACE方法从鹤望兰黄色花萼中克隆到类黄酮生物合成途径关键基因SrF3′5′H。该cDNA全长1 766 bp,具有完整的开放阅读框（ORF）,共1 509个碱基,编码503个氨基酸。氨基酸同源性分析表明,SrF3′5′H编码的氨基酸序列与已报道的其他植物的F3′5′H蛋白具有很高的同源性。系统进化树分析显示,鹤望兰SrF3′5′H与非洲紫罗兰蛋白亲缘关系较近。应用半定量PCR分析表明,SrF3′5′H在始花期转录水平达到最高,且在蓝色花瓣中表达最高,在黄色花萼中几乎没有表达。     

Synthesis and anti-HIV evaluation of new 2′,3′- dideoxy-3′-thiacytidine prodrugs

Abstract

A series of anti-HIV prodrugs possessing various polyaminated side arms have been developed. The incorporation of a N-Boc protected monoamine or diamine side arm into the backbone of the 2′,3′-dideoxy-3′-thiacytidine 1 (BCH-189) provided an increase in antiviral potency, which could be several orders magnitude greater than the parent drug (1) depending on the cell culture systems used (MT-4 or MDMs). Twenty six 2′,3′-dideoxy-3′-thiacytidine prodrugs which differ from each other by the length, the nature of the 5′-O function and the 5′-O or /and N-4 position on the nucleoside moiety were synthesized. Among this new series of prodrugs, several congeners (12c and 12a) were found to inhibit HIV-1 replication in cell culture with 50% effective concentrations ECso of 10 and 50 nM respectively, in MT-4 cells. Compound 12c was found more active on infected MDMs cells with 50% effective concentration of 0.01 nM. The synthesis and the antiviral properties of these compounds are discussed.