A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

2′5′-Phosphodiesterase Activity Studies with Xyloadenosine Analogs of 2–5A Cores

Abstract

Sequential substitution of xyloadenosine into the trimeric and tetrameric 2–5A cores¹ allows evaluation of the importance of the 3′ hydroxyl groups to 2′5′-phosphodiesterase (PDE) activity.     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Genetic mapping: Chromosomes 2–5

Summary Genetic maps of chromosomes 2 and 4 constructed from pair-wise lod score data from family studies and regional assignments for markers are presented. Two loci are mapped on chromosome 2 and multiple crossing-over is suggested as an explanation for the poor fit to the data in females. The best map of chromosome 4 gives the genetic locations of five markers with the Stoltzfus (SF) blood group distal to MNS on the long arm and GC close to the centromere on the short arm. This position for GC is outside its provisional regional assignment and possible reasons for this discrepancy are discussed. The GM-PI linkage group has a score of less than-1.0 with chromosome 4 suggesting that it may be excluded from that chromosome.The regional assignment for markers on chromosome 2–5 are also shown.     

Host-guest Interactions in Intercalates Zr(HPO4)2·2C2H5OH and VOPO4·2C2H5OH

Molecular mechanics simulations using Cerius² modelling environment combined with vibrational spectroscopy (IR and Raman) have been used to study the host-guest interactions in zirconium and vanadyl phosphate intercalated with ethanole. The strategy of investigation is based on the comparison of vibrational spectra for the host compound, intercalate and guest species. This comparison confirmed the rigidity of VOPO₄- and Zr(HPO₄)₂-layers during the intercalation and provided us with the basis for the strategy of modelling. Molecular mechanics simulations revealed the structure of intercalates and enabled to analyse the host-guest interaction energy and bonding geometry. The bilayer arrangement of ethanole molecules in the interlayer space with two differently bonded ethanole molecules has been found in both intercalates. The average interaction energy ethanole-layer for two differently bonded ethanole molecules is : 127.5 and 135.7 kcal·mol^-1 in Zr(HPO₄)₂·2C₂H₅OH, respectively 94.0 and 104.4 kcal·mol^-1 in VOPO₄·2C₂H₅OH. The Coulombic contribution to the ethanole-layer interaction energy is predominant in all cases, but the hydrogen bonding contribution is much higher in Zr(HPO₄)₂·2C₂H₅OH than in VOPO₄·2C₂H₅OH. Present results of modelling enabled the interpretation of vibrational spectra and explanation of small changes in positions and shapes of spectral bands, in infrared and Raman spectra, proceeding from the host structure to intercalates.     

Efficient Synthesis of 5-Carboxy-2′-Deoxypyrimidine Nucleoside 5′-Triphosphates

An efficient P(V)–N activation method for the synthesis of 5-carboxy-2′-deoxyuridine and 5-carboxy-2′-deoxycytidine triphosphates directly from the corresponding phosphoropiperidate precursors has been developed.     

pppA2’p5’A2’p5’A保护病毒感染细胞的普遍性

pppA2’p5’A2’p5’A(简称2’-5’P_3A_3)是干扰素作用于细胞后诱导产生的物质。干扰素的作用机理很复杂,其中之一是2’-5’寡聚腺苷酸合成酶的活力增加,此酶以ATP为底物合成2’-5’P_3A_3及其同系物2’-5’P_3An。但2’-5’P_3A_3或2’-5’P_3A_n本身是否具有抗病毒作用,干扰素的抗病毒作用是否通过2’-5’P_3A_3或2’-5’P_3An而进行,这是一个很     

pppA2′p5′A2′p5′A保护病毒感染细胞的普遍性

pppA2′p5′A2′p5′A(简称2′-5′P_3A_3)是干扰素作用于细胞后诱导产生的物质。干扰素的作用机理很复杂,其中之一是2′-5′寡聚腺苷酸合成酶的活力增加,此酶以ATP为底物合成2′-5′P_3A_3及其同系物2′-5′P_3An。但2′-5′P_3A_3或2′-5′P_3A_n本身是否具有抗病毒作用,干扰素的抗病毒作用是否通过2′-5′P_3A_3或2′-5′P_3A_n而进行,这是一个很     

类固醇5-α还原酶样基因Srd5α2l2的克隆及表达分析

采用表达序列标签（EST）介导的基因克隆和表达谱分析,从小鼠心脏克隆了一个cDNA为2 348 bp,主要在心脏表达的新基因Srd5α2l2（GenBank Acc No.AF548365）.该基因由12个外显子组成,3′非翻译区富含ATTTA序列,最大开放阅读框编码一个由361个氨基酸组成的假定蛋白,该蛋白质C端含有类固醇5-α还原酶的保守结构域（3-oxo-5-alpha-steroid 4-dehydrogenase,STEROID_DH）.生物信息学分析表明,人cDNA 克隆DKFZp313D0829（AL833108）是Srd5α2l2的人同源基因.经同源性检索,支持Srd5α2l2的全部41条EST中25条来自小鼠心脏组织.RT-PCR检测初步证实,该基因主要在心脏中表达,而在其他组织中不表达或弱表达.综合考虑Srd5α2l2的序列特征和表达谱,Srd5α2l2可能在心脏组织中发挥重要作用.     

Studies Towards the Large Scale Chemical Synthesis of the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H 4 and -2′,3′,4′,5′,5″- 2H 5

Abstract

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3′,4′,5′,5″- ²H ₄ and -2′,3′,4′,5′,5″- ²H ₅ from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-α,β-D-allofuranose-3,4-d ₂ 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 16 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ) or to 1-O-methyl-3,5-di-O-benzyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 18 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ).     

真核翻译起始因子5A2(eIF5A2)表达载体的构建与鉴定

目的:构建真核翻译起始因子5A2(eIF5A2)真核表达载体。方法:PCR扩增eIF5A2片段,通过酶切连接将目的片段定向克隆至真核表达载体pIRES2-eGFP的多克隆位点,菌落PCR、质粒PCR及DNA测序对质粒进行鉴定。结果:菌落PCR、质粒PCR及DNA测序鉴定结果均表明载体构建正确。结论:成功构建了真核表达载体,并且命名为pIRES2-5A2,为下一步eIF5A2基因在人类肿瘤中作用的研究奠定了基础。     

Time-resolved fluoroimmunoassay of 5-methyl-2′-deoxycytidine

We describe time-resolved fluoroimmunoassay of 5-methyl-2′-deoxycytidine (5MedCyd). The assay is based on the use of a highly specific antiserum raised in rabbits against BSA-conjugated 5-methylcytidine (5MeCyd). The tracer in the solid-phase time-resolved fluoroimmunoassay (TR-FIA) was antigen-selected anti-5MedCyd labeled with Europium. Thyroglobulin-linked 5MeCyd served as the solid-phase antigen. The measuring range for the fluoroimmunoassay was from less than 1 to 5000 pmol per assay of 5MedCyd. A good correlation between the results obtained with the TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in human leukemic cells and other DNA samples. TR-FIA has several advantages over the more laborious techniques available so far: (i) high sensitivity, (ii) large assay ranges, (iii) rapidity and large number of simultaneous assays, (iv) simplicity, and (v) low cost provided that the laboratory has equipment for time-resolved fluorometry.     

Nkx2—5基因与心脏发育

心脏发育是一个极其复杂的过程，它涉及胚胎发育过程中不同时间、不同空间的若干基因的先后表达。大量研究表明Ｎｋｘ２－５／Ｃｓｘ基因作为与发育密切相关的同源盒基因（ｈｏｍｅｏｂｏｘｇｅｎｅ）的一种，在心脏发育过程中起重要作用。本文根据已发表的文献资料，系统地阐述Ｎｋｘ２－５基因的结构、表达及其在心脏的调节功能与途径，进而在分子水平上对心脏的发生和发病有更深入的了解。     

Efficient Synthesis of 2′-Amino-2′-deoxypyrimidine 5′-Triphosphates

Abstract

The synthesis of 2′-amino-2′-deoxypyrimidine 5′-triphosphates is described. The 2′-amino-2′-deoxyuridine 5′-triphosphate is obtained from uridine in four steps with 25% overall yield. The 2′-amino-2′-deoxycytidine 5′-triphosphate is obtained from uridine in seven steps with 13% overall yield.     

Microbiological synthesis of 5-ethyl- and (E)-5-(2-bromovinyl)-2′-deoxyuridine

Summary The title compounds were prepared by an enzymatic transdeoxyribosylation from 2 dGuo or 2 dThd to the respective heterocyclic bases, 5-ethyluracil and (E)-5-(2-bromovinyl)uracil, using the whole bacterial cells ofEscherichia coli as a biocatalyst.     

Efficient Large-Scale Synthesis of 5′-O-Dimethoxytrityl-N 4-Benzoyl-5-Methyl-2′-Deoxycytidine

An efficient process to synthesize 5′-O-dimethoxytrityl-N⁴-benzoyl-5-methyl-2 ′-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multi-kilogram quantities for routine use in oligonucleotide therapeutics.     

Relationship Between Conformation and Antiviral Activity -IV. 5-Ethyl-2′-Deoxyurldine and 5-Ethyl-2′-Deoxycytidine +

Abstract

5-Ethyl-2′-deoxyuridine(EtdUrd), though a potent inhibitor of herpes simplex virus (HSV) replication, is rapidly catabolized to produce an inactive pyrimidine base by thymidine and/or uridine phosphorylases. 5-Ethyl-2′-deoxycytidine (EtdCyd) was synthesized to confer metabolic stability and thus improve efficacy against systemic HSV infections. EtdCyd was inactive against HSV in the presence or absence of deaminase inhibitors in VERO cells up to 2 mM. The relationship between molecular conformation and antiherpes activity for EtdUrd and EtdCyd is discussed.

     

荷叶中的生物碱及其对5-HT 2A和5-HT 2C受体的激动作用研究CSCD

对荷叶中的生物碱进行了分离、鉴定和调脂减肥活性研究。本研究结合传统酸提碱沉法与现代高效液相色谱制备技术,从荷叶中分离、纯化到11个生物碱,分别被鉴定为N-氧基原荷叶碱(1)、原荷叶碱(2)、莲碱(3)、降氧化北美黄连次碱(4)、荜茇宁(5)、巴婆碱(6)、O-去甲基荷叶碱(7)、N-去甲基荷叶碱(8)、荷叶碱(9)、衡州乌药碱(10)和亚美罂粟碱(11),其中,化合物1、4和5为首次从荷叶中分得。测试所得化合物对5-HT_(2A)和5-HT_(2C)受体的激动作用,结果表明11个生物碱对5-HT_(2A)受体均具有一定的激动作用,进一步揭示了荷叶调脂减肥的可能药效基础和作用机理。