一种新型独特芋螺毒素的分离与结构鉴定

从中国南海的一种食虫芋螺独特芋螺(Conus caracteristicus)的毒液中分离得到一种新的芋螺毒素, 对其进行了结构鉴定和化学合成. 结果表明, 这是一种新的T-超家族芋螺毒素.     

4-羟基脯氨酸立体异构对α-芋螺毒素溶液构象的影响

在α-芋螺毒素及其它家族的芋螺毒素中, 脯氨酸的羟基化是非常普遍的后转录修饰方式. 在天然芋螺毒素中脯氨酸的羟基常采用反式构型, 且该残基对芋螺毒素的结构与生物活性产生了重要的影响, 而顺式构型的羟脯氨酸对α-芋螺毒素的折叠与生物活性的影响还鲜有研究. 本工作通过二维(2D)溶液核磁共振方法测定了经过化学修饰的三个含有反式或顺式羟脯氨酸的α-芋螺毒素的溶液结构, 它们是α4/7亚家族芋螺毒素肽[γ15E]Sr1B、[O7O'/γ15E]Sr1B和[O6O'/γ14E]Vc1A. 研究表明, 羟基顺反异构化学修饰对芋螺毒素的结构影响显著. 羟脯氨酸羟基的反式到顺式修饰导致α-芋螺毒素肽明显的溶液构象变化, 这些变化包括二级结构的改变、关键残基的侧链取向变化以及氢键性质的改变. [O7O'/γ15E]Sr1B与[γ15E]Sr1B相比, 典型的α-芋螺毒素ω弯曲结构发生形变. 而[O6O'/γ14E]Vc1A不同于Vc1A的是末端转角结构的缺失. 本工作加深了对α-芋螺毒素肽的化学修饰法的理解, 该方法是阐明α-芋螺毒素结构-生物活性关系的有用工具.     

一种含两对密集二硫键的模拟肽

利用α-芋螺毒素的骨架(CC—C—C)及天然堂皇芋螺毒素的氨基酸组成,设计了一个由11个氨基酸组成的线性短肽NTLCCEGCMCY-COOH.该肽在缓冲溶液中氧化折叠后二硫键只形成一种连接方式[C(1)—C(4),C(2)—C(3)],区别于大部分的α-芋螺毒素的二硫键连接方式[C(1)—C(3),C(2)—C(4)],且该肽具有镇痛活性.这是目前合成的二硫键最密集的α-芋螺毒素样模拟肽,可作为药物分子设计的模板.     

α-芋螺毒素构效关系与分子设计

α-芋螺毒素(α-conotoxins)是从芋螺毒液中提取到的一类活性多肽. 与其它家族的芋螺毒素相比, 它们含二硫键少, 结构相对简单, 由于作用于神经肌肉接头的N-乙酰胆碱受体(nAChRs)的不同亚型, 拮抗乙酰胆碱, 可作为鉴定nAChRs亚型及其亚基的有效工具, 已成为芋螺毒素结构改造的最佳先导化合物. 利用HyperChem软件包的量子化学半经验方法AM1对8个具有代表性的α-芋螺毒素进行了量子化学计算, 研究了它们的电子结构及构效关系. 结果表明, 空间结构的相似性使它们作用于同一受体, 局部结构差异而导致的电子结构的较大差别是它们能作用于不同受体亚型的重要原因. 在此基础上, 以α-芋螺毒素GI为模型设计了7个类似物并进行了量子化学计算, 比较了类似物与GI在空间结构及电子结构方面的特征.     

新型桶形芋螺毒素BtIIIB的分离纯化、氨基酸序列测定及二硫键定位研究

采用凝胶色谱、高效液相色谱等方法从栖息于我国南海的桶形芋螺的毒液中纯化出一个新的芋螺毒素BtIIIB.采用氨基酸组成分析、质谱分析和Edman降解测定BtIIIB为15肽,其氨基酸序列为:CCELPCHGCVPCCWP.结构中含有6个半胱氨酸,形成3对分子内二硫键.采用部分还原的方法,分步还原毒素中的二硫键,用氰基化试剂衍生生成巯基,然后在碱性条件下将衍生的产物进行裂解,用MALDI-TOFMS测定裂解后片段的分子量,确定了其二硫键的配对方式为Cys1-Cys13,Cys2-Cys9,Cys6-Cys12的部分交叉式结构,是芋螺毒素中比较特别的配对方式.     

新型桶形芋螺毒素BtⅢB的分离纯化、氨基酸序列测定及二硫键定位研究

采用凝胶色谱、高效液相色谱等方法从栖息于我国南海的桶形芋螺的毒液中纯化出一个新的芋螺毒素BtⅢB.采用氨基酸组成分析、质谱分析和Ednan降解测定BtⅢB为15肽,其氨基酸序列为:CCELPCHGCVPCCWP.结构中含有6个半胱氨酸,形成3对分子内二硫键.采用部分还原的方法,分步还原毒素中的二硫键,用氰基化试剂衍生生成巯基,然后在碱性条件下将衍生的产物进行裂解,用MALDI-TOF MS测定裂解后片段的分子量,确定了其二硫键的配对方式为Cys1-Cys13,Cys2-Cys9,Cys6-Cys12的部分交叉式结构,是芋螺毒素中比较特别的配对方式.     

新型桶形芋螺毒素BtIIIB的分离纯化、氨基酸序列测定及二硫键定位研究

采用凝胶色谱、高效液相色谱等方法从栖息于我国南海的桶形芋螺的毒液中纯化出一个新的芋螺毒素BtIIIB．采用氨基酸组成分析、质谱分析和Edman降解测定BtIIIB为15肽, 其氨基酸序列为: CCELPCHGCVPCCWP．结构中含有6个半胱氨酸, 形成3对分子内二硫键．采用部分还原的方法, 分步还原毒素中的二硫键, 用氰基化试剂衍生生成巯基, 然后在碱性条件下将衍生的产物进行裂解, 用MALDI-TOF MS测定裂解后片段的分子量, 确定了其二硫键的配对方式为Cys¹-Cys¹³, Cys²-Cys⁹, Cys⁶-Cys¹²的部分交叉式结构, 是芋螺毒素中比较特别的配对方式．     

μ-芋螺毒素及其类似物的定量构效关系研究

μ-芋螺毒素是肌肉型钠离子通道的专一性阻断剂,本文主要采用PLS（PartialLeastSquare）多元数学分析方法对μ-芋螺毒素及其17个类似物进行了定量构效关系研究,建立了QSAR模型,其模型的交叉验证值R2＝0．813,Y实验值与Y预测值的相关系数0．903．计算结果表明,对分子活性影响比较大的是13位精氨酸残基和分子中的电荷变化,增加分子的正电荷,将提高分子的活性,其次是19,2,12,9,和17位氨基酸残基．     

不饱和螺环缩酮的合成及对桔小实蝇(Bactrocera dorsalis)性引诱活性与构效关系研究（英文）

螺环缩酮结构片段广泛存在于许多具有不同来源的生物活性天然产物中,该片段往往对生物活性起着重要作用.合成了两类结构新颖的螺环缩酮类化合物,并以甲基丁香酚为标准品对照,测试其对桔小实蝇(Bactrocera dorsalis)的电生理活性.结果表明:雄性和雌性桔蝇对大部分化合物小实有明显的电生理响应.螺环缩酮的立体化学和其苯环上取代基对电生理响应有一定的影响.     

生物毒素的分析

生物毒素是一类具有重要军事和生物医学应用价值的生物源化学物质。许多生物毒素可以作为临床药物或导向药物而受到广泛研究，另外又由于其具有极高的毒性，已成为重要的潜在性生化战剂，受到世界各国军事学家的关注。本文着重介绍了蓖麻毒素(Ricin)、相思子毒素(Abrin)、河豚毒素(Tetrodotoxin)、石房蛤毒素(Saxitoxin)、芋螺毒素(Conotoxin)等五种主要生物毒素的分离分析鉴定方法。     

Structure–Activity Studies of Cysteine‐Rich α‐Conotoxins that Inhibit High‐Voltage‐Activated Calcium Channels via GABAB Receptor Activation Reveal a Minimal Functional Motif

α‐Conotoxins are disulfide‐rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α‐conotoxins that also modulate voltage‐gated calcium channels by acting as G protein‐coupled GABA_B receptor (GABA_BR) agonists. These α‐conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α‐conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α‐conotoxins known to inhibit high voltage‐activated calcium channels via GABA_BR activation. Remarkably, all disulfide isomers of the active α‐conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.     

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.     

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.     

Conotoxins as research tools and drug leads

The complex mixture of biologically active peptides that constitute the venom of Conus species provides a rich source of ion channel neurotoxins. These peptides, commonly known as conotoxins, exhibit a high degree of selectivity and potency for different ion channels and their subtypes making them invaluable tools for unravelling the secrets of the nervous system. Furthermore, several conotoxin molecules have profound applications in drug discovery, with some examples currently undergoing clinical trials. Despite their relatively easy access by chemical synthesis, rapid access to libraries of conotoxin analogues for use in structure-activity relationship studies still poses a significant limitation. This is exacerbated in conotoxins containing multiple disulfide bonds, which often require synthetic strategies utilising several steps. This review will examine the structure and activity of some of the known classes of conotoxins and will highlight their potential as neuropharmacological tools and as drug leads. Some of the classical and more recent approaches to the chemical synthesis of conotoxins, particularly with respect to the controlled formation of disulfide bonds will be discussed in detail. Finally, some examples of structure-activity relationship studies will be discussed, as well as some novel approaches for designing conotoxin analogues.     

Inorganic/organic polymer coatings for textiles to realize water repellent and antimicrobial properties—A study with respect to textile comfort

SiO₂ coatings and inorganic/organic polymer hybrid coatings were applied onto textiles, and the textile properties were investigated with respect to parameters of textile comfort as stiffness, water uptake, and air permeability. Two different types of textiles (viscose and polyamide) were dip-coated with coating solutions of a pure silica sol and a polymer-modified silica sol. Only with low concentrated coating solutions a sufficient low stiffness and therefore an appropriate textile comfort could be realized. Analogously the water uptake of the treated textiles was decreased and sufficient high values were only reached with highly diluted coating solutions. Therefore, it was investigated whether such diluted coating solutions could be used for modification of textiles to add new beneficial properties. To reach hydrophobic textile properties one sol was modified with perfluorooctyltriethoxysilane. For antimicrobial functionalization a second sol was modified with silver. It was shown, for the application of new textile properties like water repellency or antimicrobial activity only concentrations ≤1% were necessary. In this case, the increase of textile stiffness was appropriate low, so the textile comfort was preserved while new functional properties were applied. Therefore, the presented diluted coating agents could be appropriate means for textile refinement and offer new textile applications. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1562–1568, 2010     

Wearable Solar Cells by Stacking Textile Electrodes

A new and general method to produce flexible, wearable dye‐sensitized solar cell (DSC) textiles by the stacking of two textile electrodes has been developed. A metal–textile electrode that was made from micrometer‐sized metal wires was used as a working electrode, while the textile counter electrode was woven from highly aligned carbon nanotube fibers with high mechanical strengths and electrical conductivities. The resulting DSC textile exhibited a high energy conversion efficiency that was well maintained under bending. Compared with the woven DSC textiles that are based on wire‐shaped devices, this stacked DSC textile unexpectedly exhibited a unique deformation from a rectangle to a parallelogram, which is highly desired in portable electronics. This lightweight and wearable stacked DSC textile is superior to conventional planar DSCs because the energy conversion efficiency of the stacked DSC textile was independent of the angle of incident light.     

Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity

Conotoxins are a large family of disulfide‐rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33‐residue conotoxin Φ‐MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ‐MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β‐hairpins that resemble the N‐terminal domain of granulin. Similar to granulin, Φ‐MiXXVIIA promotes cell proliferation (EC₅₀ 17.85 μm ) while inhibiting apoptosis (EC₅₀ 2.2 μm ). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ‐MiXXVIIA expands the repertoire of disulfide‐rich conotoxins that recognize mammalian receptors.     

The Terebridae and teretoxins: Combining phylogeny and anatomy for concerted discovery of bioactive compounds

The Conoidea superfamily, comprised of cone snails, terebrids, and turrids, is an exceptionally promising group for the discovery of natural peptide toxins. The potential of conoidean toxins has been realized with the distribution of the first Conus (cone snail) drug, Prialt (ziconotide), an analgesic used to alleviate chronic pain in HIV and cancer patients. Cone snail toxins (conotoxins) are highly variable, a consequence of a high mutation rate associated to duplication events and positive selection. As Conus and terebrids diverged in the early Paleocene, the toxins from terebrids (teretoxins) may demonstrate highly divergent and unique functionalities. Recent analyses of the Terebridae, a largely distributed family with more than 300 described species, indicate they have evolutionary and pharmacological potential. Based on a three gene (COI, 12S and 16S) molecular phylogeny, including ~50 species from the West-Pacific, five main terebrid lineages were discriminated: two of these lineages independently lost their venom apparatus, and one venomous lineage was previously unknown. Knowing the phylogenetic relationships within the Terebridae aids in effectively targeting divergent lineages with novel peptide toxins. Preliminary results indicate that teretoxins are similar in structure and composition to conotoxins, suggesting teretoxins are an attractive line of research to discover and develop new therapeutics that target ion channels and receptors. Using conotoxins as a guideline, and innovative natural products discovery strategies, such as the Concerted Discovery Strategy, the potential of the Terebridae and their toxins are explored as a pioneering pharmacological resource.     

基于铂/碳球的乙酰胆碱酯酶传感器在农药乐果检测中的应用研究

基于农药乐果对乙酰胆碱酯酶的抑制作用,构建生物传感器,实现了农药乐果的快速、高灵敏检测。合成了纳米材料铂/碳球(Pt/Cs),利用其比表面积大、导电性好的优势,构建乙酰胆碱酯酶(ACh E)传感器。铂/碳球修饰电极比裸电极的阻抗更低,峰电流增加了147.06%,说明该材料能很好地保持酶的催化活性。在最优实验条件下,用ACh E传感器检测农药乐果,在1.0×10~(-9)~1.0×10~(-6)g/L范围,乐果浓度的负对数与抑制率呈良好的线性关系,其检出限为7.3×10~(-12)g/L(按抑制率为10%计算)。对纺织品样品进行加标回收实验,测得回收率为86.2%~101.7%。