Fluorescent analysis of translesion DNA synthesis by using a novel, non-natural nucleotide analogue

The replication of damaged DNA is a promutagenic process that can lead to disease development. This report evaluates the dynamics of nucleotide incorporation opposite an abasic site, a commonly formed DNA lesion, by using two fluorescent nucleotide analogues, 2-aminopurine deoxyribose triphosphate (2-APTP) and 5-phenylindole deoxyribose triphosphate (5-PhITP). In both cases, the kinetics of incorporation were compared by using a 32P-radiolabel extension assay versus a fluorescence-quenching assay. Although 2-APTP is efficiently incorporated opposite a templating nucleobase (thymine), the kinetics for incorporation opposite an abasic site are significantly slower. The lower catalytic efficiency hinders its use as a probe to study translesion DNA synthesis. In contrast, the rate constant for the incorporation of 5-PhITP opposite the DNA lesion is 100-fold faster than that for 2-APTP. Nearly identical kinetic parameters are obtained from fluorescence quenching or the 32P-radiolabel assay. Surprisingly, distinct differences in the kinetics of 5-PhITP incorporation opposite the DNA lesion are detected when using either bacteriophage T4 DNA polymerase or the Escherichia coli Klenow fragment. These differences suggest that the dynamics of nucleotide incorporation opposite an abasic site are polymerase-dependent. Collectively, these data indicate that 5-PhITP can be used to perform real-time analyses of translesion DNA synthesis as well as to functionally probe differences in polymerase function.     

Fluorescent Nucleoside Analogs: Probes for Investigating Nucleic Acid Structure and Function

Base-modified fluorescent nucleoside analog probes have been very valuable in the study of nucleic acid structure and function. Many of them structurally resemble natural bases, and also display useful properties, such as large Stokes shifts and sensitivity to microenvironment changes. Therefore, unlike traditional fluorescence probes, which mostly report global changes, nucleoside analogs, when incorporated into oligonucleotides, can photophysically report changes that occur around the site of interest, at the nucleotide level. In this review, we provide an overview of various strategies that have been employed to design base-modified fluorescent nucleoside analogs. Then we review recent developments and applications of new generation fluorescent nucleoside analogs with a particular focus on the synthesis, photophysical characterizations and applications of heterobicycle-conjugated pyrimidine nucleoside analogs that have been developed by our group. These analogs, which have a minimal effect on the structures of the oligonucleotides into which they are incorporated, show emission in the visible region and excellent fluorescence solvatochromism. Notably, unlike the majority of fluorescent nucleoside analogs developed so far, these analogs retain their fluorescence efficiency when incorporated into oligonucleotides. We anticipate that these nucleoside analogs, with such photophysical properties, would be useful in designing robust biophysical assays to study nucleic acids.     

A Fluorescent sp2-iminosugar with pharmacological chaperone activity for gaucher disease: synthesis and intracellular distribution studies

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.     

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis,Fluorescent Properties,and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.     

Making Epothilones Fluoresce: Design,Synthesis, and Biological Characterization of a Fluorescent N12‐Aza‐Epothilone (Azathilone)

A green fluorescent 12‐aza‐epothilone (azathilone) derivative has been prepared through the attachment of the 4‐nitro‐2,1,3‐benzoxadiazole (NBD) fluorophore to the 12‐nitrogen atom of the azamacrolide core structure. While less potent than natural epothilones or different N12‐acylated azathilone derivatives, NBD‐azathilone ( 3 ) promotes tubulin assembly, inhibits cancer cell proliferation in vitro and arrests the cell cycle at the G2/M transition. Most significantly, the binding of 3 to cellular microtubules (MTs) could be directly visualized by confocal fluorescence microscopy. Based on competition binding experiments with laulimalide‐stabilized MTs in vitro, the N12‐Boc substituted azathilone 1 , Epo A, and NBD‐azathilone ( 3 ) all interact with the same tubulin‐binding site. Computational studies provided a structural model of the complexes between β‐tubulin and 1 or 3 , respectively, in which the NBD moiety of 3 or the BOC moiety of 1 directly and specifically contribute to MT binding. Collectively, these data demonstrate that the cellular effects of 3 and, by inference, also of other azathilones are the result of their interactions with the cellular MT network.     

丹磺酰胺为染料母体的Zn2+荧光探针合成及光谱性能

基于碳酸酐酶(CA)-芳香磺酰胺抑制剂相互作用的原理,本文合成了一种新的Zn2 荧光探针2-丹磺酰氨乙基-1-DPA(2-[5-[二甲基胺]1-萘磺酰氨乙基]1-N,N-二吡啶甲基胺)(1),其中Zn2 对荧光团丹磺酰胺有很好的亲和力,N,N-二(2-吡啶甲基)乙二胺作为接受体对Zn2 具有很好的选择性。在生理条件下(pH7.4),λx=334nm,λem=540nm。加入Zn2 后,λex基本不变,λem蓝移到520nm,荧光强度增大将近4倍。表观解离常数(Kd)在纳摩尔范围内,在生物应用中具有足够的敏感性。另外,多种生物体中重要的金属离子对1的荧光没有影响,如Ca2 、Mg2 等。Zn2 -1络合物的荧光强度受pH变化的影响不大,特别是在pH6-9之间影响很小。1的光物理性质表明它是一种对Zn2 有特殊选择性并有效的荧光探针。     

Design, synthesis, and evaluation of 5'-diphenyl nucleoside analogues as inhibitors of the Plasmodium falciparum dUTPase

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a potential drug target for malaria. We previously reported some 5'-tritylated deoxyuridine analogues (both cyclic and acyclic) as selective inhibitors of the Plasmodium falciparum dUTPase. Modelling studies indicated that it might be possible to replace the trityl group with a diphenyl moiety, as two of the phenyl groups are buried, whereas the third is exposed to solvent. Herein we report the synthesis and evaluation of some diphenyl analogues that have lower lipophilicity and molecular weight than the trityl lead compound. Co-crystal structures show that the diphenyl inhibitors bind in a similar manner to the corresponding trityl derivatives, with the two phenyl moieties occupying the predicted buried phenyl binding sites. The diphenyl compounds prepared show similar or slightly lower inhibition of PfdUTPase, and similar or weaker inhibition of parasite growth than the trityl compounds.     

Design,synthesis, and preliminary evaluation as antimicrobial activity of novel spiro-1, 3-thiazolidine C-acyclic nucleoside analogs

Synthesis of a new class of 1, 3-thiazolidine nucleoside analogs is described. Reaction of 2-amino-2-deoxy-D-glucopyranose hydrochloride 2 with carbon disulfide yielded 5-hydroxy-4-(D-arabino-1, 2, 3, 4-tetrahydroxybutyl)-thiazolidin-2-thione 3, which on acetylation yielded 5-acetoxy-4-(D-arabino-1, 2, 3, 4-tetraacetoxy-butyl)-thiazolidin-2-thione 4. The acetylated sugar 4 reacted with hydrazonoyl chlorides 1a–f, affording the 5-acetoxy-4-(D-arabino-1, 2, 3, 4-tetraacetoxybutyl)-spiro-1 Chen, H., Fan, Y-H., Natarajan, A., Guo, Y., Iyasere, J., Harbinski, F., Luus, L., Christ, W., Aktas, H. and Halperin, J. A. 2004. Bioorg. Med. Chem. Lett., 14: 5401–5405. [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] 3 Nasr, M. N., Gineinah, M. M. and El-Bendary, E. R. 2003. Arch. Pharm., 336: 560–566. [Crossref] , [Google Scholar]thiazolidine-2,2′ -1 Chen, H., Fan, Y-H., Natarajan, A., Guo, Y., Iyasere, J., Harbinski, F., Luus, L., Christ, W., Aktas, H. and Halperin, J. A. 2004. Bioorg. Med. Chem. Lett., 14: 5401–5405. [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] 3 Nasr, M. N., Gineinah, M. M. and El-Bendary, E. R. 2003. Arch. Pharm., 336: 560–566. [Crossref] , [Google Scholar] 4 Belleau, B., Brasilli, L., Chan, L. and Zacheri, B. 1993. Cameron J. Bioorg. Med. Chem. Lett., 3: 1723–1928. [Crossref], [Web of Science ®] , [Google Scholar]thiadiazole derivatives 8a–f. The antibacterial activity of the novel 1, 3-thiazolidine-2,2′ -spiro- 1 Chen, H., Fan, Y-H., Natarajan, A., Guo, Y., Iyasere, J., Harbinski, F., Luus, L., Christ, W., Aktas, H. and Halperin, J. A. 2004. Bioorg. Med. Chem. Lett., 14: 5401–5405. [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] 3 Nasr, M. N., Gineinah, M. M. and El-Bendary, E. R. 2003. Arch. Pharm., 336: 560–566. [Crossref] , [Google Scholar] 4 Belleau, B., Brasilli, L., Chan, L. and Zacheri, B. 1993. Cameron J. Bioorg. Med. Chem. Lett., 3: 1723–1928. [Crossref], [Web of Science ®] , [Google Scholar]thiadiazole nucleoside analogs is highlighted. All compounds with free NH group in the thiazolidine series 8a–f showed significant biological activity against all the standard strains.     

水热法制备Y2O3:Eu3+微米棒及其荧光性能表征

利用水热法制备了Y2O3和Y2O3:Eu3+,探讨了反应温度、反应时间及氢氧化钠溶液浓度对产物晶型的影响,确定了生成较好晶型的反应条件为：反应温度180℃,反应时间24 h,氢氧化钠溶液浓度2 mol/L. 研究了Y3+和Eu3+的配比对Y2O3:Eu3+荧光性能的影响. 结果表明,当n(Y3+):n(Eu3+)的比例为100:5时,其荧光强度最佳. TEM分析表明,Y2O3:Eu3+粉末具有直径约0.2~0.6 mm、长度为几到十几微米的棒状结构.     

Molecular Design of an Environmentally Sensitive Fluorescent Nucleoside, 3‐Deaza‐2′‐Deoxyadenosine Derivative: Distinguishing Thymine by Probing the DNA Minor Groove

An environmentally sensitive fluorescent nucleoside containing a 3‐deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3‐naphthylethynylated 3‐deaza‐2′‐deoxyadenosine (^3nzA, 1 ) exhibited dual fluorescence emission from an intramolecular charge‐transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength.     

Tropolone-Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism,Enzymatic Incorporation into DNA and HeLa Cell Internalization

Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future.     

Effect of a new nonoxide additive,Y3Si2C2, on the thermal conductivity and mechanical properties of Si3N4 ceramics

Enhancement of the thermal conductivity of silicon nitride is usually achieved by sacrificing its mechanical properties (bending strength). In this study, β-Si₃N₄ ceramics were prepared using self-synthesized Y₃Si₂C₂ and MgO as sintering additives. It was found that the thermal conductivity of the Si₃N₄ ceramics was remarkably improved without sacrificing their mechanical properties. The microstructure and properties of the Si₃N₄ ceramics were analyzed and compared with those of the Y₂O₃-MgO additives. The addition of Y₃Si₂C₂ eliminated the inherent SiO₂ and introduced nitrogen to increase the N/O ratio of the grain-boundary phase, inducing Si₃N₄ grain growth, increasing Si₃N₄ grain contiguity, and reducing lattice oxygen content in Si₃N₄. Therefore, by replacing Y₂O₃ with Y₃Si₂C₂, the thermal conductivity of the Si₃N₄ ceramics was significantly increased by 31.5% from 85 to 111.8Wm⁻¹K⁻¹, but the bending strength only slightly decreased from 704 ± 63MPa to 669 ± 33MPa.     

Eu~(3+)掺杂Y_2O_3-Al_2O_3-SiO_2玻璃的制备与荧光性能研究

采用高温熔融法制备了Eu3+掺杂Y2O3-Al2O3-SiO2荧光玻璃,探讨了成分对该体系玻璃形成能力的影响,并对不同Eu3+掺杂浓度下的荧光性能进行了研究.结果表明,熔融温度为1500℃条件下,SiO2含量对该体系的玻璃形成能力影响明显,Y/Al摩尔比为3/5时,SiO2含量在52%—68%(摩尔分数)范围内时可以获得玻璃.掺杂Eu3+的Y2O3-Al2O3-SiO2玻璃具有荧光性能,在395nm波长激发下,在588 nm和614 nm处出现明显的发射峰.随着Eu3+掺杂浓度的增加,该荧光玻璃的发射波长不变,但发射强度有所变化;当Eu3+掺杂浓度为1.5%(摩尔分数)时,特征发射峰强度最大.     

Endo‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement

β‐Glucoside‐configured cyclophellitols are activity‐based probes (ABPs) that allow sensitive detection of β‐glucosidases. Their applicability to detect proteins fused with β‐glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M‐777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4‐methylumbelliferyl β‐d ‐lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre‐blocking with conduritol β‐epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β‐glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high‐resolution detection moieties) should assist further research in living cells and organisms.     

Novel and Selective Fluorescent σ2‐Receptor Ligand with a 3,4‐Dihydroisoquinolin‐1‐one Scaffold: A Tool to Study σ2 Receptors in Living Cells

Although sigma‐2 (σ₂) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a σ₂‐selective fluorescent tracer (compound 5 ) as a specific tool to study σ₂ receptors. By using flow cytometry with 5 , we performed competition binding studies on three different cell lines where we also detected the content of the σ₂ receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed σ₁/σ₂ fluorescent tracer ( 1 ) also allowed for the detection of σ₁ receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for σ₁, and Scatchard analysis for σ₂ receptors). Thus, we have produced powerful new tools for research on the σ whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of σ receptors in oncology.     

纳米Y_2O_2S:Eu~(3+)红色荧光粉的间接法制备及发光性质

用间接法制备纳米级红色荧光粉Y2O2S:Eu3+(物质的量之比为Eu3+/Y3+=0.02)。XRD测试表明,制得纳米级的Y2O2S:Eu3+为单一纯相。透射电子显微镜(TEM)扫描结果显示纳米材料的平均颗粒尺寸为30 nm,与Debye-Scherre公式计算的结果基本一致。荧光光谱中,激发光谱分为3个区域,200-280 nm的区域为Y2O2S基质的激子激发带,峰值为262 nm;280-380 nm的区域属于Eu3+→O2-(S2-)的电荷迁移带,峰值为319 nm;而位于399 nm和472 nm的线状光谱则属于Eu3+的4 f组态内部电子之间的跃迁。