硫代磷酸酯寡聚核苷酸的立体控制合成研究进展

硫代磷酸酯(PS)修饰的寡聚核苷酸被广泛应用于生物化学机制研究、生物医药和新材料等领域.硫代磷酸酯中心P原子的立体构型对于PS核酸的生化性质具有显著影响.因此,高效、高立体选择性地合成PS寡聚核苷酸在过去的30年中引起了广泛的关注.本文分类归纳了制备立体纯PS寡聚核苷酸的方法,重点总结了近10年来基于立体纯单体和手性催化剂进行立体控制合成PS寡聚核苷酸的重要进展,对不同合成方法的优缺点进行了对比分析,最后对立体纯PS寡聚核苷酸合成的前景进行了展望.     

氰基修饰的新型寡聚噻吩衍生物的合成

以2,3,4,5-四碘代噻吩为起始原料,利用Suzuki偶联反应来构筑分子骨架,合成了3个氰基和烷基修饰的新型寡聚噻吩衍生物,其结构经1H NMR,13C NMR,EI-MS和元素分析表征。     

聚苯胺纳米线修饰的DNA电化学传感器的研究

采用电流密度递减的方法在玻碳电极表面修饰聚苯胺纳米线(PAINW), 以SEM对其形貌进行表征, 测得PAINW的尖端直径在80～100 nm之间. 以乙基-(3-二甲基丙基)碳化二亚胺盐酸盐(EDC)为偶联活化剂, 将5'-磷酸基修饰的寡聚核苷酸片断共价固定在聚苯胺修饰的电极上, 一定条件下, 以亚甲基蓝为电化学杂交指示剂, 采用差分脉冲伏安法对杂交信号进行检测, 实现了对特定序列DNA片段的互补、非互补序列的识别.     

基于硫化镉纳米团簇标记DNA电化学传感的研究

合成了表面具有自由羧基的硫化镉纳米团簇，以乙基-（3-二甲基丙基）碳二 亚胺盐酸盐为偶联活化剂，将其标记于人工合成的5'端氨基修饰的寡聚核苷酸片段 上，制备成CdS纳米团簇标记DNA探针，该寡聚核苷酸片段与大肠杆菌肠毒素基因相 关。在一定的条件下，使基与固定晨玻碳电极表面的待测DNA序列进行杂交反应， 利用阳极溶出示差脉冲伏安法（ASDPV）间接测定Cd的量，实现对互补、非互补 DNA片段的识别和电化学检测，从而对大肠杆菌肠毒素基因片段识别和检测。     

富G寡聚核苷酸修饰的金纳米粒子与细胞相互作用的研究

通过Au-S键将两种链长一致、序列不同的富鸟嘌呤(G)单链寡聚核苷酸PolyG_1和PolyG_2分别修饰到13 nm金纳米粒子(GNPs)表面,合成了两种单链寡聚核苷酸(ssDNAs)与GNPs复合物(PolyG_1-GNPs和PolyG_2-GNPs)。所合成的PolyG_1-GNPs和PolyG_2-GNPs在复杂分散介质中具有良好的胶体稳定性。采用紫外-可见吸收光谱、细胞透射电镜和电感耦合等离子体质谱等分析方法,考察ss DNA序列对PolyG_1-GNPs和PolyG_2-GNPs与细胞相互作用的影响。结果表明,PolyG_1-GNPs和PolyG_2-GNPs均具有较低的细胞毒性,并表现与能量相关的细胞内吞行为,ssDNA的序列决定PolyG_1-GNPs和PolyG_2-GNPs的细胞吞噬量及其在细胞内的分散状态,具有G4二级结构的PolyG_2能够显著增加细胞对其所修饰的GNPs的吞噬量和GNPs在细胞中的稳定性。     

锆离子改善十烷基磷酸组装膜结构及性能研究

利用烷基磷酸、金属锆离子和不锈钢电极表面的相互作用,通过表面自组装,在304不锈钢电极表面分别修饰了单层和双层烷基磷酸自组装膜,采用接触角和极化曲线对修饰电极进行测量.实验表明,双层烷基磷酸修饰膜具有更佳的耐腐蚀性能.     

基于纳米金胶标记DNA探针的电化学DNA传感器研究

以纳米金胶为标记物,将其标记于人工合成的5-端巯基修饰的寡聚核苷酸片段上,制成了具有电化学活性的金胶标记DNA电化学探针;在一定条件下,使其与固定在玻碳电极表面的靶序列进行杂交反应,利用ssDNA与其互补链杂交的高度序列选择性和极强的分子识别能力,以及纳米金胶的电化学活性,实现对特定序列DNA片段的电化学检测以及对DNA碱基突变的识别.     

聚氧乙烯功能基复合修饰聚氨酯的合成及其血液相容性研究 (Ⅰ)──聚氨酯-接枝-十八烷基聚氧乙烯

通过十八烷基聚氧乙烯和环氧氯丙烷的封端反应制备了α-环氧基-ω-十八烷基聚氧乙烯大单体.并采用BF₃·Et₂O引发THF和大单体共聚合,得到了梳状的十八烷基聚氧乙烯接枝共聚醚.以该共聚醚为软段合成了十八烷基和聚氧乙烯复合修饰的聚氨酯(PEU-g-PEO-C₁₈).通过血小板粘附试验对材料的体外抗凝血性实验结果表明,采用具有选择性吸附白蛋白功能的十八烷基和PEO复合修饰聚氨酯,材料表面血小板粘附量明显减少.材料血液相容性的改善可能来源于疏水性的十八烷基和亲水性聚氧乙烯的协同作用.     

2-氨基-5-烷基-1,3,4-噻二唑修饰环糊精的制备与表征

在N2气保护下, 用单-(6-对甲苯磺酰基)-CD(β-CD-6-OTs)和过量的2-氨基-5-烷基-1,3,4-噻二唑在80 ℃反应2 d, 合成了5种新的2-氨基-5-烷基-1,3,4-噻二唑修饰β-环糊精. 化合物的结构用IR, 1H NMR, 13C NMR, UV, MS和元素分析等方法进行了表征. 由于环糊精的屏蔽效应, 在2-氨基-1,3,4-噻二唑修饰β-环糊精的 1H NMR中, 修饰产物中的噻二唑质子发生了高场位移. 其它的谱图数据同理论值相吻合, 这证明合成与分离方法是可行的. 研究了产物的生物活性, 结果显示部分化合物(2d, 2e)的抗菌活性明显增强.     

噻吩修饰的新型并五苯衍生物的合成及其光谱性能

通过在并五苯的6-位和13-位引入寡聚噻吩取代基,成功地合成了三个噻吩修饰的并五苯衍生物(1a～lc),其结构经NMR,IR,MS和元素分析表征,其中1b和lc未见文献报道.并研究了1的甲苯溶液和固体薄膜的UV性能.     

Synthesis and properties of oligonucleotides containing C8-deoxyguanosine arylamine adducts of borderline carcinogens

C8-Arylamine-dG adducts of borderline carcinogens and the bladder and breast carcinogen 4-aminobiphenyl were prepared using cross-coupling chemistry. These adducts were converted into the corresponding C8-arylamine-5'-O-DMTr-2'-deoxyguanosine phosphoramidites and then used as building blocks for automated synthesis of site-specifically modified oligonucleotides. The oligonucleotides were characterized by UV melting temperature analysis, enzymatic digestion, and circular dichroism.     

Photocaging strategy for functionalisation of oligonucleotides and its applications for oligonucleotide labelling and cyclisation

We have used a photocaging strategy to develop novel phosphoramidites and expand the repertoire of protecting groups for modification of oligonucleotides by solid-phase synthesis. We synthesised five photolabile phosphoramidites and four new photolabile controlled pore glasses (CPGs). By using these photolabile phosphoramidites and CPGs, modified oligodeoxynucleotides (ODNs) with phosphate, amine, acid, thiol and carbonyl moieties at 5' and/or 3' ends were readily synthesised. To the best of our knowledge, this is the first report of introducing a carbonyl at the 5' end and thiol groups at both ends of ODNs with photolabile modifiers. Terminal labelling was also easily realised in solution or by on-column solid-phase synthesis. By using the photolabile amine modifier and the photolabile acid CPG, cyclisation of an oligodeoxynucleotide was achieved with good yields. This study provides an alternative way to introduce functional groups into oligonucleotides and expand the scope of oligonucleotide bio-orthogonal labelling.     

Phosphoramidite building blocks for efficient incorporation of 2′-O-aminoethoxy(and propoxy)methyl nucleosides into oligonucleotides

A simple and efficient method for the preparation of eight phosphoramidite building blocks for incorporation of 2′-O-(2-aminoethoxymethyl)ribonucleosides and 2′-O-(3-aminopropoxymethyl)ribonucleosides into synthetic oligonucleotides has been developed. The synthetic routes are maximally convergent and provide sufficient amounts of phosphoramidites for several solid-phase synthesis coupling reactions. Using acyclic derivatives 17a,b the overall yields of phosphoramidites 2 and 3 were increased up to 50% for pyrimidine nucleosides and up to 30% for purine derivatives with substantial decrease of total reaction steps. The 2′-O-substituent was found to be stable during oligonucleotide synthesis. The resulting oligonucleotides are of particular interest for post-synthetic functionalization and conjugation.     

Synthesis of Photolabile 5′‐O‐Phosphoramidites for the Photolithographic Production of Microarrays of Inversely Oriented Oligonucleotides

The photolabile 3′‐O‐{[2‐(2‐nitrophenyl)propoxy]carbonyl}‐protected 5′‐phosphoramidites ( 16 – 18 ) were synthesized (see Scheme) for an alternative mode of light‐directed production of oligonucleotide arrays. Because of the characteristics of these monomeric building blocks, photolithographic in situ DNA synthesis occurred in 5′→3′ direction, in agreement with the orientation of enzymatic synthesis. Synthesis yields were as good as those of conventional reactions. The resulting oligonucleotides are attached to the surface via their 5′‐termini, while the 3′‐hydroxy groups are available as substrates for enzymatic reactions such as primer extension upon hybridization of a DNA template (see Fig. 2). The production of such oligonucleotide chips adds new procedural avenues to the growing number of applications of DNA microarrays.     

3'-nitrophenylpropyloxycarbonyl (NPPOC) protecting groups for high-fidelity automated 5' --> 3' photochemical DNA synthesis

[structure: see text]. The most powerful DNA microarrays would be prepared by photolithography with free 3'-ends that could be processed enzymatically. A photoremovable group that could be removed in quantitative yield would ensure high purity of the synthesized probes. We have developed new pyrimidine building blocks for 5' --> 3' DNA synthesis with high cycle yields using the NPPOC (3'-nitrophenylpropyloxycarbonyl) protecting group. These phosphoramidites were proved in automated photochemical DNA synthesis on a modified synthesizer.     

Dialkynylpyrenes: strongly fluorescent, environment-sensitive DNA building blocks

Two isomeric dialkynylpyrene phosphoramidites and their incorporation into oligonucleotides are described. The pyrene units closely resemble the well-known perylene bisimide dye PDI with regard to the ability to self-organize within a DNA duplex. In addition, dialkynylpyrenes exhibit significant monomer and remarkably strong excimer fluorescence. The dialkynylpyrene building blocks are promising candidates for applications in diagnostic tools, such as excimer-based molecular beacons, as well as for novel DNA-based materials with special optical properties.     

Nucleotides,Part LXIX,Synthesis of Phosphoramidite Building Blocks of Isoxanthopterin N8‐(2′‐Deoxy‐β‐D‐ribonucleosides): New Fluorescence Markers for Oligonucleotide Synthesis

The chemical synthesis of isoxanthopterin and 6‐phenylisoxanthopterin N⁸‐(2′‐deoxy‐β‐D ‐ribofuranosyl nucleosides) is described as well as their conversion into suitably protected 3′‐phosphoramidite building blocks to be used as marker molecules for DNA synthesis. Applying the npe/npeoc (=2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl) strategy, we used the new building blocks in the preparation of oligonucleotides by an automated solid‐support approach. The hybridization properties of a series of labelled oligomers were studied by UV‐melting techniques. It was found that the newly synthesized markers only slightly interfered with the abilities of the labelled oligomers to form stable duplexes with complementary oligonucleotides.     

New strategies for cyclization and bicyclization of oligonucleotides by click chemistry assisted by microwaves

The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide-alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new phosphoramidite building blocks and new solid supports were designed to introduce alkyne and bromo functions into the same oligonucleotide by solid-phase synthesis on a DNA synthesizer. The bromine atom was then substituted by sodium azide to yield azide oligonucleotides. Cyclizations were found to be more efficient in solution than on solid support. This method allowed the efficient preparation of cyclic (6- to 20-mers), branched (with one or two dangling sequences), and bicyclic (2 x 10-mers) oligonucleotides.     

Accurate molecular weight measurements of nucleoside phosphoramidites: a suitable matrix of mass spectrometry

Nucleoside phosphoramidites (PAs) are the most widely used building blocks in contemporary solid-phase synthesis of oligonucleotides. The accurate molecular weight (MW) measurements of such molecules, which contain acid-labile moieties, may be easily determined by mass spectrometry using a matrix system, triethanolamine (TEOA)-NaCl, on liquid secondary ion mass spectrometry (LSIMS) equipped with a double-focusing mass spectrometer. The present method measures rapidly and easily the accurate MWs of various PAs as adduct ions [M+Na]⁺ with average mass error smaller than 0.4 ppm, allowing the formulas of various PAs in place of elemental analysis. Further, it was found that intensities of molecular-related ions could be enhanced to the highest degree by adjustment of the mole ratio of PA and NaCl fixing the amount of TEOA on LSIMS, making the present method powerful tool for the MS identification of PAs.     

Enhanced stereoselectivity in internucleotidic bond formation by the use of the chiral ribose moiety of thymidine

This paper deals with the synthesis of new cyclic thymidine 3'-phosphoramidite building blocks having a covalent linker between the trityl type 5'-hydroxyl protecting group and the phosphorus atom attached to the 3'-hydroxyl group of thymidine. The ring structures were designed to reduce the conformational freedom around the phosphorus center so that the stereoselectivity in the internucleotide linkage formation would be improved. The linkers were also designed to be removed readily by treatment with aqueous ammonia. These building blocks were synthesized in good yield by one-pot cyclization of the diol precursors with dichloro(N,N-diisopropylamino)phosphine, despite their large-membered ring. Various activators having 1H-tetrazole, imidazole, and triazole structures were investigated to find the best selectivity in the synthesis of thymidylyl(5'-3')thymidine phosphorothioate. It turned out that our cyclic phosphoramidites gave preferentially the R(p) diastereoisomer in high coupling yield applicable to the solid-phase synthesis of oligodeoxynucleotides. It should be noted that high stereoselectivity was achieved without any chiral sources other than the 2'-deoxyribose moiety itself. The mechanistic studies revealed the importance of the steric bulk and the acidity of the activators. It was also found that the steric bulk of the alcoholic nucleophile was an important factor that determined the stereoselectivity in our systems.