Structures And Biological Activity Of Alkaloids And Other Natural Products

两栖类皮肤提供了广泛的生物活性生物碱，其中许多具有独特的药理活性和治疗潜力。这些生物碱包括有效的钠通道激活剂巴曲霉毒素、非竞争性烟碱受体通道阻滞剂组氨酸毒素、pumiliotoxins/allopumiliotoxins/homopumiliotoxins及其类似物，其中一些由于对钠通道的作用而具有强肌和强心活性，以及依巴替丁，这是一种非常有效和选择性的尼古丁激动剂，具有很强的抗伤害感受活性。其他生物碱包括十氢喹啉类、吡咯利齐定类、吲哚里西定类、喹唑类、来咪嗪类，以及各种三环生物碱，包括螺吡咯里西定肟、地理毒素、伪菲咯烷类、环戊喹啉类和球果桂类。有机化合物的结构鉴定现在几乎完全基于光谱分析，使用紫外线(UV)、红外(气相IR)、质量(MS)和核磁共振(NMR)光谱技术。我们的天然产品计划依赖于强大的分离和光谱技术的开发，用于分析从两栖动物皮肤、节肢动物和其他来源的提取物中获得的复杂混合物中微量存在的生物碱和其他化合物。关键技术是气相色谱(GC)或高效液相色谱(HPLC)分离，然后对UV、IR、MS以及有望的核磁共振数据进行在线分析。随着甲醛和甲酸的GC分析中顺式二醇的氢化、酰化、丁基硼化和N-甲基化等微观化学反应的发展，这些技术已经对青蛙皮提取物中代表约26个结构类别的800多种生物碱进行了详细的表征。高效液相色谱-质谱法可以研究所有生物碱，即使是那些高分子量或高极性的生物碱，但由于缺乏大气压化学电离(APCI)或电喷雾电离(ESI)的广泛碎裂，只能提供有限的结构见解。使用电子碰撞电离(EIMS)的GC-MS分析提供了丰富的、可诊断的碎裂模式，而化学电离(CIMS)则提供了分子量和可交换的OH和NH基团的数量。这种开创性的光谱研究已经扩展到在碰撞激活的CIMS模式下开发和应用串联质谱仪，展示和阐明不同于传统EIMS的碎片，以及对传统EIMS的补充。气相GC-FTIR(傅立叶变换红外光谱)的分析潜力使其能够从传统的IR用途(鉴定官能团，如羟基、羰基、双键和三键等)扩展到提供有价值的立体化学见解(顺环或跨环连接，Bohlmann带分析，以指示氢在氮附近碳上的取向等)。我们小组去年应用了一种新的FTIR方法，称为甲基计数法，它允许在红外光谱中进行详细的CH积分，大大提高了我们的结构解析能力。手性GC分析已经用合成样品建立了许多生物碱的绝对立体化学。GC-MS和GC-FTIR，在某些情况下，结合详细的核磁共振分析，甚至用于结构验证的合成，已经描绘了400多个生物碱的结构。用微探针进行的核磁共振分析目前已应用于少数仅10微克的生物碱样品。目前从中南美洲和马达加斯加的两栖动物和节肢动物的提取物中鉴定出约100种新的生物碱，其中一些代表新的结构类别，包括N-甲基十氢喹啉、二烷胺和脱氢叠氮碱。某些灰甲虫被发现含有蝙蝠毒素，似乎是毒镖蛙和某些鸟类中发现的蝙蝠毒素的饮食来源。作为多种两栖类皮肤生物碱的食物来源的螨类、蚂蚁、甲虫和千足虫已被鉴定出来，特别是甲螨和许多具有分支碳骨架的齐丁类，桃金娘蚂蚁用于其他具有直线型碳骨架的齐丁类，甲虫用于三环球孢子碱生物碱，千足虫用于螺吡咯烷酮。此外，还从结构上定义了蚂蚁中的新生物碱。蜘蛛蛾(Eudulophasia)幼虫从植物(Spigelia)中截留兰尼定的作用已被发现。使用高效液相色谱-紫外-质谱仪重新检测了一种古老的黑蟾皮提取物，发现至少有5种蟾酥二烯内酯和3种万寿菊内酯。这一结果与最初的生物测定结果一致，即哇巴因提取物抑制哇巴因与心肌功能所必需的Na/K-ATP酶结合。在此，负离子质谱仪和在高效液相色谱溶剂中使用重水对部分结构的归属是有用的。其中一种毒素PTX 251D对蚊子和火蚁具有对映体选择性接触毒性。两栖生物碱的主要生物靶标似乎既是电压敏感的离子通道，又是配体门控的离子通道，特别是钠、钙和烟碱通道。某些毒素被发现激活伤害性感觉通路，可能是通过与钠通道的相互作用。 这个项目本身就是被终止的。如果发现新的方法和技术，允许使用仍可从青蛙皮中提取的许多微量提取物进行化学分析和生物分析，它可能会在以后重新浮出水面。我们的实验室将保留这些样品和相关文件。

A wide range of biologically active alkaloids, many of which have unique profiles of pharmacological activity and therapeutic potential, has been provided by amphibian skin. These alkaloids include batrachotoxins, which are potent activators of sodium channels, histrionicotoxins, which are noncompetitive blockers of nicotinic receptor-channels, pumiliotoxins/allopumiliotoxins/homopumiliotoxins and related congeners, some of which have myotonic and cardiotonic activity due to effects on sodium channels and epibatidine, an extremely potent and selective nicotinic agonist with potent antinociceptive activity. Further alkaloids include decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines, lehmizidines, and a variety of tricyclic alkaloids, including spiropyrrolizidine oximes, gephyrotoxins, pseudophrynamines, cyclopentaquinolizidines and coccinellines. Structure elucidation of organic compounds is now based almost exclusively on spectroscopic analysis, using ultraviolet (UV), infrared (vapor-phase IR), mass (MS), and nuclear magnetic resonance (NMR) spectral techniques. Our natural products program has relied on the development of powerful separation and spectral techniques for the analysis of alkaloids and other compounds present in minute amounts in complex mixtures obtained in extracts from amphibian skin, arthropods, and other sources. The key techniques are gas chromatographic (GC) or high performance liquid chromatographic (HPLC) separation, followed by analysis online of UV, IR, MS and hopefully, NMR data. These techniques, along with development of microchemical reactions including hydrogenation, acylation, butylboronation of cis-diols and N-methylation on GC analysis with formaldehyde and formic acid, have been responsible for the detailed characterization of over 800 alkaloids, representing some 26 structural classes in frog skin extracts. HPLC-MS allows study of all alkaloids, even those of high molecular weight or polarity that do not GC, but gives only limited structural insights because of lack of extensive fragmentation with either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). GC-MS analysis using electron impact ionization (EIMS) provides rich, diagnostic patterns of fragmentation, while chemical ionization (CIMS) provides molecular weight and, with deuterated ammonia, the number of exchangeable OH and NH groups. Such pioneering spectroscopic research has been extended to developing and applying tandem mass spectrometry in the collision-activated CIMS mode, demonstrating and elucidating fragmentations different from and complementary to conventional EIMS. The analytical potential of vapor-phase GC-FTIR (Fourier transform IR) has allowed extension from traditional uses of IR (identification of functional groups like OH, carbonyl, double and triple bonds, etc.), to providing valuable stereochemical insights (cis- or trans-ring junctions, Bohlmann band analysis to indicate orientation of hydrogens on carbons adjacent to nitrogen, etc.). A new method in FTIR, applied during the last year by our group and still being developed by us, named methyl counting, allows a detailed CH integration in the IR spectrum, greatly enhancing our ability in the process of structure elucidation. Chiral GC analysis has established with synthetic samples the absolute stereochemistry of many alkaloids. GC-MS and GC-FTIR, in conjunction in some cases with detailed NMR analysis and even synthesis for structural verification, have delineated structures of over 400 alkaloids. NMR analysis with microprobe has now been applied to a few alkaloid samples of only 10 ug. Current extracts from amphibians and arthropods of Central and South America and Madagascar have led to identification of about 100 new alkaloids, some representing new structural classes, including N-methyldecahydroquinolines, dialkylamines and dehydroizidines. Certain melyrid beetles were found to contain batrachotoxins and appear likely to be the dietary source of batrachotoxins found in poison dart frogs and certain birds. The mites, ants, beetles and millipedes that are dietary sources of many classes of amphibian skin alkaloids have been identified, notably oribatid mites for pumiliotoxins and many izidines with branched carbon skeletons, myrmicine ants for other izidines with linear carbon skeletons, beetles for the tricyclic coccinelline alkaloids and siphonotid millipedes for the spiropyrrolizidines. Further novel alkaloids from ants have been structurally defined. The sequestration of ryanodine from plants (Spigelia) by larvae of the spider moth (Eudulophasia) has been discovered. The use of HPLC-UV-MS to re-examine an old extract of a Melanophryniscus toad skin has revealed at least 5 bufadienolides and 3 cardenolides. This result is consistent with the original bioassay, the inhibition by that extract of ouabain-binding to a Na/K-ATP-ase enzyme, essential in heart muscle function. Here, negative-ion MS and use of deuterated water in the HPLC solvent were useful in assigning partial structures. One of the pumiliotoxins, namely PTX 251D, had enantioselective contact toxicity for mosquitoes and fire ants. The major biological targets for the amphibian alkaloids appear to be both voltage-sensitive and ligand-gated ion channels, in particular sodium, calcium and nicotinic channels. Certain pumiliotoxins were found to activate nociceptive sensory pathways, presumably through interaction with sodium channels. This project as such is being terminated. It may resurface later if new methodology and techniques are discovered that allow the chemical analysis and also the biological analysis to be done with the many trace amount extracts still available from frog skin. Our lab will keep those samples and related documentation.