化学修饰的寡核苷酸: 合成、性质和应用

本文综述了近年来化学修饰的寡核苷酸研究的进展,介绍了化学修饰的寡核苷酸的种类, 化学合成方法, 生物作用原理及应用方面已达到的成継34This review deals with the progress in the research of chemically modified oligonucleotides: the synthesis, the chemical and biological properties and the potential applications as a new research tool and new therapeutic approach.     

Complexation of well-controlled low-molecular weight polyelectrolytes with antisense oligonucleotides

The influences of polymer-related properties such as molecular weight, charge density, counter ion, and hydrophilic block on the complexation of polyelectrolytes and a fluorescein-labeled oligonucleotide (ON) were investigated. A series of well-defined and well-controlled 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA) polymers and block copolymers were prepared using living anionic and radical polymerization methods. Fluorescence measurement was used to reveal the effects of polymer molecular weight, charge density, and counter ion type on the complexation. PolyDMAEMA samples having double molecular weights of the chosen oligonucleotide gave the optimal complexation performance. Kinetic studies showed that high-molecular weight/high-charge density polymer samples produced very stable complexes. The fully charged polyDMAEMA displayed the strongest binding with the ON. These complexes were therefore less sensitive to the changes in the environment. PolyDMAEMA–DMSQ samples had slightly higher complexation ability than polyDMAEMA–MCQ (DMSQ: dimethylsulfate quat; MCQ: methylchloride quat). Both poly(DMAEMA-b-HEMA) and poly(DMAEMA–MCQ-b-PEG) block copolymers showed good complexation ability and steric stability [HEMA: 2-hydroxyethyl methacrylate; PEG: poly(ethylene glycol)]. PEG, but not HEMA block, enhanced the effectiveness of polyDMAEMA–MCQ binding with the ON.     

Chemical composition of the volatile oil from Cynanchum stauntonii and its activities of anti-influenza virus

The volatile oil of the roots of Cynanchum stauntonii was examined by gas chromatography–mass spectrometry (GC–MS). Thirty-eight constituents were identified. (E,E)-2,4-Decadienal, 3-efhyl-4-methypentanol, 5-pentyl-3H-furan-2-one, (E,Z)-2,4-decadienal and 2(3H)-furanone,dihydro-5-pentyl were found to be the major components. The volatile oil exhibited the activities against influenza virus in vitro (IC₅₀s = 64 μg/ml). In in vivo experiment, it prevented influenza virus-induced deaths in a dose-dependent manner.     

TiO2对流感病毒(H1N1)灭活作用的研究

利用滴度测定和透射电镜观察研究了365 nm的紫外光照射下TiO2对流感病毒(H1N1)的灭活性能, 并结合催化剂样品的XRD分析、 N2气吸附性能测定及其在实验条件下的表面Zeta电势的测量结果, 探讨了催化剂用量、 焙烧温度、 比表面积以及表面电性与灭活性能的关系. 研究结果表明, 400 ℃时焙烧的TiO2对H1N1的灭活性最好; TiO2的表面电性对灭活性有显著影响; TiO2对H1N1的光催化灭活作用首先发生在H1N1的纤突部分, 纤突部分的破坏导致H1N1的失活, 分解直至矿化.      

Characterization of complexes formed by polypropylene imine dendrimers and anti-HIV oligonucleotides

Current anti-HIV therapies are capable of controlling viral infection but do not represent a definitive cure. They rely on the administration of antiretroviral nucleoside analogues, either alone or in combination with vectors. Dendrimers are branched, synthetic polymers with layered architectures, promising non-viral vectors in gene therapy. The aim of the paper was to study the interactions between three anti-HIV antisense oligonucleotides (ODNs): SREV, ANTI TAR, GEM91 and different generation polypropylene imine dendrimers (PPI) by monitoring changes in the fluorescence polarization of fluorescein attached to the ends of the ODNs when increasing concentrations of dendrimers were added. Laser Doppler electrophoresis, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to characterize, respectively, zeta potential, particle size and morphology of dendriplexes formed in different molar ratios. Antisense oligonucleotides interacted with polypropylene imine dendrimers in different molar ratios depending on generation. Zeta potential of dendriplexes varied from (-25 to -21) mV to -5 mV (for PPIG3 and PPIG4 complexes) and to zero (for PPIG2 complexes). The structures presented a polydisperse size from about 50 nm to even 700-800 nm by TEM and about 250 nm by DLS. It means that besides single dendriplexes, aggregates were also present.     

Phosphoramidites and solid supports based on N-substituted 2,4-dihydroxybutyramides: universal reagents for synthesis of modified oligonucleotides

A general and convenient method for synthesis of modified oligonucleotides by use of new non-nucleoside phosphoramidites is reported. A chiral 1,3-diol backbone of the modifying reagents is generated either from (R)-(+)-α-hydroxy-γ-butyrolactone or (R)-(−)-pantolactone. Aliphatic amines were acylated with the lactones to give the corresponding N-substituted 2,4-dihydroxybutyramides. After protection of a side chain, if necessary, the diols were converted into phosphoramidites or solid supports suitable for use in oligonucleotide synthesis. The reagents allow single, multiple or combined introduction of various functions (e.g., alkylamine, imidazole and pyrene residues) into synthetic oligonucleotides. The structures of the conjugates were confirmed by MALDI-TOF mass spectrometry.     

单管高灵敏度等温扩增技术快速检测甲型H1N1流感病毒

建立了单管逆转录环介导等温扩增法(RT-LAMP)快速检测甲型H1N1流感病毒的方法.针对甲型H1N1流感病毒的M基因和HA基因的保守区,设计了两组特异性引物,分别用于筛选甲型流感病毒及鉴定甲型H1N1流感病毒.对反应体系中的关键因素进行优化,反应结果可直接通过浊度或者SYBR Green Ⅰ荧光进行判定.本方法最低可...     

Palladium-catalyzed carbonylative annulation of terminal alkynes: synthesis of coumarins and 2-quinolones

o-Iodophenols and o-iodoaniline derivatives react with terminal alkynes under 1 atm of CO in the presence of pyridine and catalytic amounts of Pd(OAc)₂ to generate coumarins and 2-quinolones, respectively, as the only products. Terminal alkynes bearing alkyl, aryl, silyl, hydroxyl, ester and cyano substituents are effective in these processes affording the desired products in moderate yields. The formation of coumarins and 2-quinolones in this process is in stark contrast with all previously described palladium-catalyzed reactions of o-iodophenols or o-iodoanilines with terminal alkynes and CO, which have afforded chromones and 4-quinolones. Moreover, under our reaction conditions terminal alkynes insert into the carbonpalladium bond instead of undergoing a Sonogashira-type coupling as confirmed by an isotope labeling experiment.     

Design,synthesis and antimicrobial activities of 1,2,3-triazole derivatives

Fifteen 1-(4-substituted phenyl)-4-(4-bromophenyl)-5-(halo-o-hydroxyphenyl)imino-1,2,3-triazoles were designed and synthesized based on rational combination of 1,2,3-triazoles and(halo)o-hydroxyphenyl group according to the superposition principle of reinforcement of biological activities.All the compounds were tested to an in vitro antimicrobial screening against M.a.and E.c..Compounds IIe-IIo exhibited more potent antimicrobial activities against M.a.and E.c.than triclosan and fluconazole,which provided valuable information to further study of novel antimicrobial research.     

Substituent and solvent effects in the 1,3-dipolar cycloadditions for synthesis of anti-influenza agent peramivir and its analog

Influenza remains a health problem to humans. Peramivir is a FDA approved anti-influenza drug targeting the virus neuraminidase. The (3 + 2) cycloaddition reaction of 2-ethylbutanenitrile oxide with the cyclopentene dipolarophile derived from Vince lactam is a key step in the conventional synthesis of peramivir. Our study showed that conducting the (3 + 2) cycloaddition reactions with either aliphatic or aromatic nitrile oxide in hexane solution provided high percentage of the desired regioisomer, and the N-substituent having electron-withdrawing property is also beneficial to the regioselectivity. This study also demonstrated an alternative synthetic pathway of (−)-peramivir and the analog having a phenyl group in place of the 3-pentyl moiety.     

Design, synthesis, and incorporation of fluorous 5-methylcytosines into oligonucleotides

A palladium-catalyzed Negishi coupling reaction has been developed to synthesize fluorous 5-methylcytosines. These fluorous nucleosides are incorporated into the oligonucleotides that correspond to part of the promoter region of Oct4, a master gene that undergoes dynamic DNA demethylation during cellular reprogramming. The separation of the fluorous oligonucleotides from its nonfluorous analogues has been achieved through solid-phase extraction over fluorous silica, suggesting its potential use in probing DNA demethylation.     

Interdigitated array microelectrode based impedance immunosensor for detection of avian influenza virus H5N1

Continuous outbreaks of avian influenza (AI) in recent years with increasing threat to animals and human health have warranted the urgent need for rapid detection of pathogenic AI viruses. In this study, an impedance immunosensor based on an interdigitated array (IDA) microelectrode was developed as a new application for sensitive, specific and rapid detection of avian influenza virus H5N1. Polyclonal antibodies against AI virus H5N1 surface antigen HA (Hemagglutinin) were oriented on the gold microelectrode surface through protein A. Target H5N1 viruses were then captured by the immobilized antibody, resulting in a change in the impedance of the IDA microelectrode surface. Red blood cells (RBCs) were used as biolabels for further amplification of the binding reaction of the antibody-antigen (virus). The binding of target AI H5N1 onto the antibody-modified IDA microelectrode surface was further confirmed by atomic force microscopy. The impedance immunosensor could detect the target AI H5N1 virus at a titer higher than 10³ EID₅₀/ml (EID₅₀: 50% Egg Infective Dose) within 2 h. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear range for the titer of H5N1 virus was found between 10³ and 10⁷ EID₅₀/ml. Equivalent circuit analysis indicated that the electron transfer resistance of the redox probe [Fe(CN)₆]^3−/4− and the double layer capacitance were responsible for the impedance change due to the protein A modification, antibody immobilization, BSA (bovine serum albumin) blocking, H5N1 viruses binding and RBCs amplification. No significant interference was observed from non-target RNA viruses such as Newcastle disease virus and Infectious Bronchitis disease virus. (The H5N1 used in the study was inactivated virus.)     

Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1

As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (V_T) and field-effect mobility. The changes of I_D–V_G curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5 × 10⁻⁹ g mL⁻¹ to 5 × 10⁻⁶ g mL⁻¹ with a detection limit of 0.8 × 10⁻¹⁰ g mL⁻¹. Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs can be promising candidates for the development of label-free biosensors.     

Design,enantioselective synthesis,and antiviral activities against potato virus Y of axially chiral thiazine derivatives

A series of novel thiazine derivatives featuring axial chirality in both (R) and (S) configurations were successfully synthesized by N-heterocyclic carbene (NHC)-catalyzed enantioselective [3 + 3] annulations, and their potential as anti-plant virus agents against potato virus Y (PVY) was evaluated. Biological activity results demonstrated that most of these chiral thiazine derivatives exhibited significant activities against PVY. Notably, compound (S) -3g displayed a remarkable 58% inactivation effect against PVY at a concentration of 500 μg/mL, slightly surpassing the effectiveness of Ningnanmycin (NNM) at 57%. Additionally, (S) -3g exhibited curative activity of 57%, which is superior to NNM (53%). Molecular docking studies revealed preliminary insights into the distinct biological properties of the two different enantiomers, (R) or (S) -3g against PVY, wherein single enantiomer (S) -3g formed a more stable interaction with PVY-CP, as indicated by its lower binding free energy (−41.18 kcal/mol) compared to (R)- 3g (−36.9 kcal/mol). The findings in this study with a new class of axially chiral thiazine derivatives shall inspire further development of chiral heterocycles as potential drug candidates for the protection of plant virus infections.     

Functional tuning of nucleic acids by chemical modifications: tailored oligonucleotides as drugs,devices, and diagnostics

Chemical modifications of nucleic acids present vast opportunities for extending the functions and properties of these biomolecules. In general, efforts invested in this direction pertain to the introduction of reactive functional groups for further derivatizations of oligonucleotides with numerous reporter groups and for equipping nucleic acids with catalytic chemical moieties. This review deals with representative chemical modifications in the nucleobases, sugars, and the phosphate ester backbone and their application from novel catalytic RNA selection to nucleic acid-based biosensors.     

Design,Synthesis and Biological Activity of Novel Chalcone Derivatives as Anti-influenza Agents

A series of novel chalcone derivatives was designed and synthesized via a suitable synthetic strategy in good yields using commercially available 2-amino-4-nitrophenol as an initiator. The structures of the target compounds were confirmed by means of ¹H NMR, ¹³C NMR and high-resolution mass spectrometry(HRMS). And the ability of the target compounds to inhibit influenza viruses was evaluated. These compounds showed moderate inhibitory activity against influenza A(H9N2 and H5N1) viruses. Within this series, compounds S14 and S15 with good potency(IC₅₀=40.3-51.5μmol/L) could be used as lead compounds in the future.     

A simple and effective approach to the synthesis of alkynyl selenides from terminal alkynes

Alkynyl selenides were prepared under very mild conditions by reacting terminal alkynes with respective diorganic diselenides in the presence of potassium t-butoxide.The advantages of this protocol include the use of readily available substrates and reagent and good yield of the products.     

A versatile reagent for the synthesis of 5′-phosphorylated, 5′-thiophosphorylated or 5′-phosphoramidate-conjugated oligonucleotides

We report the synthesis of a new phosphorylating reagent that is easily accessible and allows not only the chemical synthesis of 5′-phosphorylated and 5′-thiophosphorylated oligonucleotides but also the 5′-conjugation through a phosphoramidate linkage. 5′-Amino-linker and 5′-alkyne oligonucleotides were obtained and the latter was conjugated by a 1,3-dipolar cycloaddition (click chemistry) with a galactosylated azide derivative to afford 5′-galactosyl oligonucleotide with high efficiency.     

meso-meso-Linked corrole dimers with modified cores: synthesis, characterization, and properties

The synthesis and characterization of the first examples of core-modified corrole dimers linked through the meso positions are described. The dimers are obtained by a simple Ag(I)- or Fe(III)-catalyzed coupling reaction in near-quantitative yields. The corroles obtained are metalated with metal ions, such as Cu(II) and Ni(II). The electronic-absorption spectral studies reveal weak electronic interaction between the two subunits and the exciton coupling observed for the free-base corrole dimer (1717 cm(-1)) is lower than that for the corresponding protonated derivative (4081 cm(-1)). The solution-state structure derived from (1)H and two-dimensional NMR spectral studies reveals a noncoplanar arrangement of two corrole units. Geometry optimization at the B3LYP-631G level also confirms the noncoplanar arrangement of corroles with a dihedral angle of 64.8 degrees between two corrole planes. The electron paramagnetic resonance (EPR) and magnetic characterization studies on the paramagnetic copper-metalated dimer indicate that both copper ions behave as independent spins without any noticeable interaction. Results of fluorescence studies reveal a bathochromic shift of about 60 nm upon dimerization. The first hyperpolarizability (beta) measured by using the hyper Rayleigh scattering (HRS) method reveals doubling of the beta values on progressing from monomer to dimer, attributed to enhanced pi conjugation. The use of copper dimers in the photocleavage of DNA is also explored. It is shown that the bimetallic copper dimer selectively cleaves the nucleic acids without affecting the proteins, suggesting a possible application of the copper complex in the removal of nucleic acid contaminants from protein extracts through a simple photolytic pathway.