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.     

用固定化5′-磷酸二酯酶工业生产5′-核苷酸

自从固定化氨基酰化酶在日本获得工业应用以来,已引起各国广泛注意,至今已有近10个固定化酶在一些先进国家投入工业使用。我们自1970年开展固定化酶研究以来,已将我     

用4,4′-二羧酸-2,2′-二喹啉测定红细胞膜蛋白

细胞膜蛋白质的测定在生物物理、生物化学及医学等领域的研究中起重要作用,许多细胞膜上酶、离子、脂类、基团及功能的测定均与膜蛋白的水平有关,需测定膜蛋白量。目前膜蛋白的测定大多采用酚试剂法,该法灵敏度高,但酚试剂在碱性溶液中不稳定,干扰因素多,特异性差,在有非离子型表面活性剂存在下易形成难溶性沉淀,不能用于自动分析仪器。     

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.     

New Strategies for Chemical Synthesis of Phosphorodithioates Derived from 2′- or 3′- or 5′-Thionucleosides

Abstract

Various phophorodithioates derived from thionucleosides were synthesis by the reaction anhydronucleosides with phosphorodithioic acids     

Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

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.     

麦芽根中5′-磷酸二酯酶和5′-磷酸单酯酶的提取

建立一种高效快速的从麦芽根中提取5′-磷酸二酯酶和磷酸单酯酶的方法。通过将麦芽根粉碎后,取一定粒度的麦芽根加一定量的去离子水浸泡,过滤后得粗酶液。用紫外分光光度法测定5′-磷酸二酯酶和5′-磷酸单酯酶的酶活,研究粉碎粒度、料液比、抽提温度、抽提时间等因素对麦芽根中5′-磷酸二酯酶和5′-磷酸单酯酶提取的影响。将原料粉碎至100目,以去离子水为提取剂,于4℃提取20h,料液质量比为1:10,5′-磷酸二酯酶的酶活力可达160U/ml。该法简单、快速、准确、适应性强,为5′-磷酸二酯酶的提取与检测提供了有效的工具。     

SYNTHESIS OF 5-(2,2′-BIPYRIDINYL AND 2,2′-BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: PRECURSORS TO METALLO-DNA CONJUGATES

ABSTRACT

The synthesis of 2,2′-bipyridinyl-2′-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2′-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2′-deoxyuridine nucleoside (Bipy²⁺-dU) with an ethylenyl linker. The Bipy²⁺-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

5,5′-联硫-2,2′-双硝基苯甲酸及其有关化合物对乙酰胆碱酯酶的别构效应

5,5'-联硫-2,2'-双硝基苯甲酸(DTNB)及其有关化合物TNB、TNB-Tch都是黄姑鱼肌肉乙酰胆碱酯酶的别构效应剂: 1.TNB非竞争性地抑制、而DTNB则激活该酶水解乙酰硫代胆碱和丁酰硫代胆碱的活力。对水解乙酰胆碱及α-萘酚乙酯的活力皆无影响。2.TNB-Tch比TNB具有更强的抑制作用。且对该酶水解乙酰胆碱、乙酰硫代胆碱及α-萘酚乙酯的活力皆有抑制作用。抑制类型与作用底物有关。底物在外周的结合能解除其抑制。3.钙离子可以解除TNB和TNB-Tch的抑制作用,而箭毒亦能与之对抗。对蛇毒乙酰胆碱酯酶,除了极低浓度(<10μM)DTNB和TNB有微弱的抑制作用外,TNB-Tch以及较高浓度的DTNB和TNB都具有强烈的激活作用。而对猪脑尾核乙酰胆碱酯酶的活力皆无影响。     

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。     

5,5′-联硫-2,2′-双硝基苯甲酸及其有关化合物对乙酰胆碱酯酶的别构效应

5,5'-联硫-2,2'-双硝基苯甲酸~*(DTNB)及其有关化合物TNB~(**)、TNB-Tch~(**)都是黄姑鱼肌肉乙酰胆碱酯酶的别构效应剂:1.TNB 非竞争性地抑制、而DTNB 则激活该酶水解乙酰硫代胆碱和丁酰硫代胆碱的活力。对水解乙酰胆碱及α-萘酚乙酯的活力皆无影响。2.TNB-Tch 比TNB 具有更强的抑制作用。且对该酶水解乙酰胆碱、乙酰硫代胆碱及α-萘酚乙酯的活力皆有抑制作用。抑制类型与作用底物有关。底物在外周的结合能解除其抑制。3.钙离子可以解除TNB 和TNB-Tch 的抑制作用,而箭毒亦能与之对抗。对蛇毒乙酰胆碱酯酶,除了极低浓度(<10 μM)DTNB 和TNB 有微弱的抑制作用外,TNB-Tch 以及较高浓度的DTNB 和TNB 都具有强烈的激活作用。而对猪脑尾核乙酰胆碱酯酶的活力皆无影响。     

Structure Sensitivity of Amino Proton Exchange in 2′ - and 5′ - Guanosine Monophosphate Dianions

Abstract

Proton NMR line broadening methods were used to determine the rates of amino proton exchange for disordered 2′ - and 5′ - GMP dianions in aqueous solutions containing tetramethylammonium (TMA⁺) cations. Replacing TMA⁺ with Na⁺ does not substantially alter the exchange rates, provided that H-bonded, Na⁺-directed tetramer structures are absent. Activation enthalpies (kcal/mol) and entropies (eu) for 2′ - GMP are: ΔH^# = 18.5 ± 1.3, ΔS^# = 9.6 ± 4.2 for theTMA⁺ salt atpH 8.10, and ΔH^# = 14.7 ± 2.6, ΔS^# = -3.7 ± 8.0 for the Na⁺ salt at pH 8.11. Extrapolated values of pseudo first-order rate constants at 25° Care in the range of k = 1–10 sec^?1. At suitable concentrations and temperatures, the Na⁺ salts of both 2′ - and 5′ - GMP formed stacked and unstacked tetramer units. Relative to the exchange kinetics observed for the disordered nucleotide, the exchange process in the tetramer units was catalyzed in half the amino protons and inhibited in the other half. The catalytic process (k < 10³ sec^?3) has been attributed to amino protons not involved in interbase H-bonding, where as the inhibited process (k > 10^?1 sec^?1) was assigned to those protons which do form such bonds. The structure-catalyzed process in both the stacked and unstacked tetramers was manifested by a loss of NMR amino proton intensity due to weighted time-averaging with the resonance for bulk water. A bridging water molecule between an amino proton and a phosphate on an adjacent nucleotide in the tetramer unit may provide a mechanistic pathway for the structure-catalyzed process.     

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。     

天然产物紫檀茋及(E)-3,5,3′-三甲氧基-4′-羟基二苯乙烯的合成

以3,5-二羟基苯乙酮为起始原料,经甲基化反应、Willgerodt-Kindler重排得到3,5-二甲氧基苯乙酸(4),化合物4分别与对羟基苯甲醛以及香兰素发生Perkin缩合反应得到E-2,3-二芳基丙烯酸(7a,7b)。化合物7a,7b分别经脱羧-异构化反应得到天然产物紫檀茋(8a)及(E)-3,5,3′-三甲氧基-4′-羟基二苯乙烯(8b),总收率分别为48.7%和43.8%。其化学结构经IR、EI-MS、1HNMR确证。     

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.