Structure Elucidation Of Alkaloids

有机化合物的结构解析现在几乎完全基于光谱分析，使用紫外（UV）、红外（IR）、质谱（MS）和核磁共振（NMR）光谱技术。我们的天然产品项目依赖于强大的光谱技术的发展，用于分析从两栖动物皮肤和其他来源提取物中获得的复杂混合物中微量的生物碱和其他化合物。由于对采集的限制和/或自然栖息地的破坏，往往无法获得更多的材料。关键技术是气相色谱（GC）或高效液相色谱（HPLC）分离，然后在线分析UV，IR和MS数据。这些技术，沿着微化学反应的发展（氘交换、氢化、酰化、顺式二醇的丁基硼化、用甲醛进行GC分析的还原性N-甲基化和其他微反应），已经负责对超过500种生物碱进行详细表征，代表青蛙皮肤提取物中的20多种结构类别。HPLC是最通用的分离工具，允许研究所有生物碱，即使是那些没有GC的高分子量或极性的生物碱，但由于缺乏大气压化学电离（APCI）或电喷雾电离（ESI）的广泛碎片化，这两种方法主要用于HPLC-MS分析，因此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、诊断性的碎片模式，而化学电离（CIMS）提供了分子量，并且使用氘代氨提供了可交换的OH和NH基团的数量。这种开创性的光谱研究已扩展到开发和应用串联质谱的碰撞激活CIMS模式，展示和阐明不同于传统的EIMS和互补的碎片。这是因为，在碰撞激活的CIMSn模式中，质子化的分子离子碎片仅释放中性分子，以产生稳定的阳离子，而EIMS的自由基分子离子碎片也产生自由基阳离子，因此基于不同的过程，产生的光谱完全不同。本部分已经并正在发展这些方法在生物碱研究中的碎裂规则和应用。气相GC-FTIR（傅里叶变换红外）的分析潜力允许从红外的传统用途（识别官能团，如OH、羰基、双键和三键等）扩展，获得立体化学的见解（顺式或反式环连接，使用Bohlmann谱带分析信息来确定氢在与氮相邻的碳上的取向等）。在这方面，开创性的研究（只有有限数量的这些仪器在世界上使用）的意义，某些红外波段和应用气相色谱-傅里叶变换红外光谱的结构测定生物碱已在我们的部分在过去几年中，从而更新的兴趣，红外光谱，最古老的技术之一。这种光谱和微量化学技术分析复杂的混合物，结合详细的核磁共振分析，当它是可行的，以分离亚毫克量的生物碱，导致超过300种生物碱的青蛙皮肤提取物的鉴定，与部分表征超过100个额外的，许多仍然代表新的结构类别。在一些情况下，合成无论是在我们的部门或合作，已被要求确认结构和生物研究提供足够的材料。对小型节肢动物提取物的分析已经确定了约30种生物碱，为蚂蚁、甲虫和千足虫作为膳食来源提供了证据。

Structure elucidation of organic compounds is now based almost exclusively on spectroscopic analysis, using ultraviolet (UV), infrared (IR), mass (MS), and nuclear magnetic resonance (NMR) spectral techniques. Our natural products program has relied on the development of powerful spectral techniques for the analysis of alkaloids and other compounds present in minute amounts in complex mixtures obtained in extracts from amphibian skin and other sources. Because of restrictions on collecting and/or destruction of natural habitat, further material often cannot be obtained. The key techniques are gas chromatographic (GC) or high performance liquid chromatographic (HPLC) separation, followed by analysis online of UV, IR and MS data. These techniques, along with development of microchemical reactions (deuterium exchange, hydrogenation, acylation, butylboronation of cis-diols, reductive N-methylation on GC analysis with formaldehyde, and other microreactions) have been responsible for the detailed characterization of over 500 alkaloids, representing more than 20 structural classes in frog skin extracts. HPLC is the most general separation tool, allowing study of all alkaloids, even those of high molecular weight or polarity that do not GC, but giving only limited structural insights because of lack of extensive fragmentation with either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), the two methods mostly used for HPLC-MS analysis. 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. This is so because, in the collision-activated CIMSn mode the protonated molecular ion fragments releasing neutral molecules only, to produce stable cations, while the radical molecular ion of EIMS fragments to also produce radical cations, and thus the spectra produced are totally different, based on different processes. Rules of fragmentation and applications of these methods to the study of alkaloids have been and are being developed in our Section. The analytical potential of vapor-phase GC-FTIR (Fourier transform IR) has allowed extension from traditional uses of IR (to identify functional groups like OH, carbonyl, double and triple bonds, etc.), to the obtaining of stereochemical insights (cis- or trans-ring junctions, use of Bohlmann band analysis information as to orientation of hydrogens on carbons adjacent to nitrogen, etc.). In this regard, pioneering studies (only a limited number of these instruments are in use in the world) on the significance of certain IR bands and application of GC-FTIR to the structure determination of alkaloids have been developed in our Section in the last few years, thus renewing interest in IR spectroscopy, one of the oldest techniques available. Such spectroscopic and microchemical techniques on analysis of complex mixtures, in conjunction with detailed NMR analysis, when it was feasible to isolate submilligram quantities of an alkaloid, have led to identification of over 300 alkaloids from frog skin extracts, with partial characterization of over 100 additional ones, many still representing new structural classes. In several cases, synthesis either in our Section or in collaboration, has been required to confirm structures and provide sufficient material for biological investigation. Analyses of extracts of small arthropods, have identified some 30 of these alkaloids, providing evidence for ants, beetles and millipedes as dietary sources.