基于点击化学反应的免疫荧光检测方法的建立和应用

本研究介绍了一种新型免疫荧光标记方法及其在细胞荧光检测中的应用技术．首先,合成了两个关键的化合物6-叠氮-己酸琥珀酰亚胺活性酯和4-乙炔基-N-乙基-1,8-萘酰亚胺,并将合成的6-叠氮-己酸琥珀酰亚胺活性酯与抗her2抗体Anti-HP15的游离氨基偶联获得叠氮化IgG,随后通过铜离子催化4-乙炔基-N-乙基-1,8-萘酰亚胺中的炔基与标记抗体的叠氮基团进行点击化学反应,同时以NHS-FITC和NHS-Rhodamine标记的抗体为阳性对照,确认了采用该标记方法的灵敏度,其检测限可达0.1 μg,EC50与阳性对照相当．然后在细胞水平进行染色分析,结果表明,叠氮标记抗体可有效应用于免疫荧光染色分析．最后,采用激光共聚焦三通道复合荧光分析法对不同标记方法及其对应的免疫荧光显色方法进行研究,确认了采用本研究开发的方法标记的抗体可与其他免疫荧光技术同时使用,且结果互不干扰．本研究通过开发一种新型的抗体标记技术,建立了一种新的免疫荧光抗体分析方法,并在细胞水平上进行了应用验证,丰富了免疫荧光抗体检测手段,该方法在未来的免疫研究中具备发展潜力和广泛应用前景．     

Second-generation difluorinated cyclooctynes for copper-free click chemistry

The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed "click chemistry", is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists.     

Starburst Dendrimers: Molecular-Level Control of Size,Shape, Surface Chemistry,Topology, and Flexibility from Atoms to Macroscopic Matter

Starburst dendrimers are three-dimensional, highly ordered oligomeric and polymeric compounds formed by reiterative reaction sequences starting from smaller molecules—“initiator cores” such as ammonia or pentaerythritol. Protecting group strategies are crucial in these syntheses, which proceed via discrete “Aufbau” stages referred to as generations. Critical molecular design parameters (CMDPs) such as size, shape, and surface chemistry may be controlled by the reactions and synthetic building blocks used. Starburst dendrimers can mimic certain properties of micelles and liposomes and even those of biomolecules and the still more complicated, but highly organized, building blocks of biological systems. Numerous applications of these compounds are conceivable, particularly in mimicking the functions of large biomolecules as drug carriers and immunogens. This new branch of “supramolecular chemistry” should spark new developments in both organic and macromolecular chemistry.     

Biometallorganische Chemie: Ein faszinierendes Forschungsgebiet

Bioorganometallic Chemistry is a new research area in which organometallic compounds are coupled with biomolecules (sugars, peptides, DNA and its constituents, steroides, vitamines, enzymes). In a narrow sense such organometallic complexes occur in nature (vitamin B₁₂), have a biological function (e.g. nickel enzymes in bacterias) or are of potential medical use (as novel drug or as marker for biomolecules). In a wider sense bioorganometallic chemistry includes simply metal complexes which besides organometallic ligands (e.g. CO, π‐hydrocarbon) have a biomolecule as ligand (e.g. with interesting structures, for catalysis).     

Sugar amino acid based scaffolds--novel peptidomimetics and their potential in combinatorial synthesis

To meet the growing demands for the development of new molecular entities for discovering new drugs and materials, organic chemists have started looking for new concepts to supplement traditional approaches. In one such approach, the expertise gained over the years in the area of organic synthesis and the rational drug-design concepts are combined together to create "nature-like" and yet unnatural organic molecules that are expected to provide leads in discovering new molecules. Emulating the basic principles followed by nature to build its vast repertoire of biomolecules, organic chemists are developing many novel multifunctional building blocks. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl groups provide an excellent opportunity for organic chemists to create structural diversities akin to nature's molecular arsenal. Recent advances in the area of combinatorial chemistry give unprecedented technological support for rapid compilations of sugar amino acid-based libraries exploiting the diversities of carbohydrate molecules and well-developed solid-phase peptide synthesis methods. This review chronicles the development of sugar amino acids as a novel class of peptidomimetic building blocks and their applications in generating desired secondary structures in peptides as well as in creating mimics of natural biopolymers.     

S66: A Well-balanced Database of Benchmark Interaction Energies Relevant to Biomolecular Structures

With numerous new quantum chemistry methods being developed in recent years and the promise of even more new methods to be developed in the near future, it is clearly critical that highly accurate, well-balanced, reference data for many different atomic and molecular properties be available for the parametrization and validation of these methods. One area of research that is of particular importance in many areas of chemistry, biology, and material science is the study of noncovalent interactions. Because these interactions are often strongly influenced by correlation effects, it is necessary to use computationally expensive high-order wave function methods to describe them accurately. Here, we present a large new database of interaction energies calculated using an accurate CCSD(T)/CBS scheme. Data are presented for 66 molecular complexes, at their reference equilibrium geometries and at 8 points systematically exploring their dissociation curves; in total, the database contains 594 points: 66 at equilibrium geometries, and 528 in dissociation curves. The data set is designed to cover the most common types of noncovalent interactions in biomolecules, while keeping a balanced representation of dispersion and electrostatic contributions. The data set is therefore well suited for testing and development of methods applicable to bioorganic systems. In addition to the benchmark CCSD(T) results, we also provide decompositions of the interaction energies by means of DFT-SAPT calculations. The data set was used to test several correlated QM methods, including those parametrized specifically for noncovalent interactions. Among these, the SCS-MI-CCSD method outperforms all other tested methods, with a root-mean-square error of 0.08 kcal/mol for the S66 data set.     

Conducting polythiophenes with a broad spectrum of reactive groups

The development of reliable and reproducible chemistries for the immobilization of biomolecules to a conducting polymer is a key challenge in the design and preparation of a CP‐based biosensor. In this article, the syntheses and electropolymerization of a series of new 3‐alkylthiophene derivatives functionalized with the most used reactive groups in immobilization chemistry, including maleimide, azide, and anhydride, are described. Despite the nucleophilic or electrophilic nature of the reactive groups, the synthesized thiophene monomers exhibit rather good polymerizability, and the reactive groups withstand the polymerization conditions and are correctly incorporated into the resulting electroactive polymers. The reactivity of the pendant reactive groups of the resulting polymers to attach biomolecules has been examined with different redox‐active, photoactive compounds as well as recognition elements as model compounds. It has been confirmed that with well‐established procedures developed for the immobilization of enzymes, the model compounds can be easily and selectively bound onto these new conducting polymers without the loss of their optical and electrochemical activity. Therefore, these conductive materials with a broad spectrum of reactive groups will provide a useful platform for developing CP‐based biosensors for a wide range of applications. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4547–4558, 2005     

Catalytic biofunctional membranes containing site-specifically immobilized enzyme arrays: a review

Biofunctional membranes normally involve the random immobilization of biomolecules to porous, polymeric membranes, often through the numerous lysine residues on the protein. In this process, bioactivity is significantly decreased largely due to different orientations of the biomolecule with respect to the membrane or to multiple point attachment. To circumvent this difficulty, while still taking advantage of the immobilization of biomolecules, site-specific immobilization of the biomolecule with the active (or binding) site directed away from the membrane is essential. In this review, we summarize our efforts involving biophysical and bioanalytical chemistry and chemical engineering, together with molecular biology, to develop and characterize such site-specifically membrane immobilized catalytic enzyme bioreactors. Site-directed mutagenesis, gene fusion technology, and post-translational modification methods are employed to effectuate the site-specific membrane immobilization. Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems. Biofunctional membranes incorporating site-specifically immobilized biomolecules provide greater efficiency of biocatalysis, bioseparations, and bioanalysis.     

SPE and purification of DNA using magnetic particles

Superparamagnetic particles have been attractive for molecular diagnostics and analytical chemistry applications due to their unique magnetic properties and their ability to interact with various biomolecules of interest. This paper presents a critical overview of magnetic nano ‐ and microparticles used as a solid phase for extraction and purification of DNAs. The mechanisms of DNA binding to the surface of functionalised magnetic particles are described. The most widely used materials including silica supports, organic polymers and other materials, mostly containing magnetite or paramagnetic metallic elements are reviewed. The main application areas of magnetic particles for DNA separation are briefly described.     

Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules

The chemical labeling of biomolecules continues to be an important tool for the study of their function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts. In addition, with the advances in biocompatible synthetic organic chemistry, a whole new field of opportunity has opened up, affording high diversity in the nature of the label as well as a choice of ligation reactions. In recent years, several different two-step labeling strategies have emerged. These rely on the introduction of a bioorthogonal attachment site into a biomolecule, then ligation of a reporter molecule to this site using bioorthogonal organic chemistry. This Perspective focuses on these techniques, their implications and future directions.     

Sugar amino acids and related molecules: Some recent developments

To meet the growing demands for the development of new molecular entities for discovering new drugs and materials, organic chemists have started working on many new concepts that can help to assimilate knowledge-based structural diversities more efficiently than ever before. Emulating the basic principles followed by Nature to build its vast repertoire of biomolecules, organic chemists are developing many novel multifunctional building blocks and using them to create ‘nature-like’ and yet unnatural organic molecules. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to Nature’s molecular arsenal. In recent years, sugar amino acids have been used extensively in the area of peptidomimetic studies. Advances made in the area of combinatorial chemistry can provide the necessary technological support for rapid compilations of sugar amino acidbased libraries exploiting the diversities of their carbohydrate frameworks and well-developed solid-phase peptide synthesis methods. This perspective article chronicles some of the recent applications of various sugar amino acids, furan amino acids, pyrrole amino acids etc. and many other related building blocks in wide-ranging peptidomimetic studies     

A novel heterobifunctional linker for facile access to bioconjugates

A convenient synthesis of a novel heterobifunctional linker molecule is described. The linker contains a thiol-reactive nitropyridyl disulfide group (Npys) and an aldehyde-reactive aminooxy group with a propensity to form disulfide and oxime linkages. The utility of the linker molecule to cross-link different biomolecules has been demonstrated by employing it in the efficient preparation of a peptide-oligonucleotide conjugate. The linker reported herein could be a useful tool for cross-coupling of different but appropriately functionalised biomolecules.     

Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry

Bioorthogonal chemistry refers to chemical reactions that can occur within a living system without altering native biochemical processes. Applications of this concept extend to studies on a group of biomolecules that includes glycans, proteins, and lipids. In this study, a strategy for isolating cell surface glycoproteins and based on bioorthogonal chemistry was employed to identify new cancer-related glycoproteins. A novel alkyne reagent containing one disulfide bond was synthesized for the enrichment of glycoproteins metabolized with peracetylated N-azidoacetylmannosamine, which was applied on three different cancer cell lines, and all isolated proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry. The strategy of purifying cell surface glycoproteins introduced in this article was shown to be reliable, and a total of 56 cell surface glycoproteins were identified. Neuronal cell adhesion molecule was found uniquely expressed in A549 lung adenocarcinoma, and its expression in non-small-cell lung carcinomas was detected by immunohistochemistry. Furthermore, a significant increase of neuronal cell adhesion molecule expression was identified in non-small-cell lung adenocarcinoma compared with adjacent noncancerous tissues, and could be a novel potential target and marker in cancer treatment and detection.     

Synthesis and post-synthetic derivatization of a cyanine-based amino acid. Application to the preparation of a novel water-soluble NIR dye

An efficient synthesis of a new fluorescent amino acid derived from a pentamethylene cyanine dye is described. Selective derivatization of its amino group with an original trisulfonated linker has led to a novel water-soluble NIR dye suitable for covalent labeling of biomolecules     

Two-dimensional nonlinear optical activity spectroscopy of coupled multi-chromophore system

Most biomolecules are chiral. A variety of optical activity measurement techniques have been extensively used to study chiral natures of complicated biological molecules such as proteins and nucleic acids. Recently, coherent two-dimensional (2D) spectroscopic techniques have been developed and widely used to study structures and dynamics of biomolecules via measuring couplings between chromophores. However, such 2D optical spectroscopic methods utilizing linearly polarized beams do not provide information on the molecular chirality. Thus, we have theoretically shown that novel 2D optical activity measurement techniques based on three- and four-wave-mixing schemes are of use to obtain the 2D spectrum of a chiral molecule. Particularly, we carried out numerical simulations of 2D optical activity spectra of polypeptides and a light-harvesting complex. These methods utilizing circularly polarized beams and related spectroscopic techniques will be of great use in understanding and elucidating the underlying mechanisms of ultrafast chemical and conformational changes of chiral biomolecules in the future.     

Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds

A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10-tetraazacyclododecane (cyclen) with one equivalent of para-functionalized alkyl 2-bromophenyl-acetate and three equivalents of tert-butyl 2-bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr3-octreotate by chemoselective oxime ligation and CuI-catalyzed azide-alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.     

天然产物化学研究的挑战和机遇

本文论述了目前我国天然产物化学研究中所存在的问题以及面临的挑战和机遇,同时就如何促进我国天然产物的新结构、新功能及其合成化学等方面的原始性创新研究和相应的对策进行了评述。     

High resolution magic angle spinning NMR in combinatorial chemistry

Solid phase organic chemistry coupled with combinatorial methods promises to increase dramatically the diversity and number of small molecules available for medical and biological applications. However, optimizing the reaction conditions can be a time consuming step, especially since analytical tools to monitor reaction progress and detect impurities for solid phase chemistry are less developed than for solution chemistry. The use of high resolution magic angle spinning (HRMAS) NMR is described here as such an analytical tool. Whereas initial applications of molecular identification using deuterated organic solvents to swell the resins presented a significant gain in time over the cleave-and-analysis methods, the introduction of a differential diffusion filter has made immediate recording of spectra possible without any sample treatment. The applications of HRMAS NMR to different solid supports that are used in combinatorial chemistry will be described in terms of rapidity, robustness and sensitivity.     

氧化铁磁性纳米粒子的制备、表面修饰及在分离和分析中的应用

氧化铁磁性纳米粒子通过表面化学修饰得到无机、有机或聚合物壳包覆在其表面。其中的壳结构既具有生物适应性,又具有可键合生物分子如细胞、蛋白质、酶、抗体和核酸的活性基团,而核具有磁性特性。本文总结了氧化铁磁性纳米粒子的制备方法,介绍了其表面化学修饰及在分离和分析应用的最新进展。     

Metathesis polymerization-derived chromatographic supports

The synthesis and properties of metathesis polymerization-derived supports for solid-phase extraction (SPE), for the on-line extraction of metal ions, ion-chromatography, reversed-phase (RP-) chromatography, and chiral chromatography is described. In addition, the metathesis polymerization-based manufacture and derivatization of monolithic supports and their use in the separation of biomolecules such as oligonucleotides, thiooligonucleotides, double-stranded DNA (dsDNA) and proteins will be summarized. Special consideration will be given to important aspects of polymer chemistry and their relevance to the properties of these new supports.