Applying computer-assisted structure elucidation algorithms for the purpose of structure validation: revisiting the NMR assignments of hexacyclinol

Computer-assisted structure elucidation (CASE) using a combination of 1D and 2D NMR data has been available for a number of years. These algorithms can be considered as "logic machines" capable of deriving all plausible structures from a set of structural constraints or "axioms", defined by the spectroscopic data and associated chemical information or prior knowledge. CASE programs allow the spectroscopist not only to determine structures from spectroscopic data but also to study the dependence of the proposed structure on changes to the set of axioms. In this article, we describe the application of the ACD/Structure Elucidator expert system to help resolve the conflict between two different hypothetical hexacyclinol structures derived by different researchers from the NMR spectra of this complex natural product. It has been shown that the combination of algorithms for both structure elucidation and structure validation delivered by the expert system enables the identification of the most probable structure as well as the associated chemical shift assignments.     

Limitations in the deduction of carbon NMR spectra from the f1 dimension of standard 2D heteronuclear experiments when applied to natural products

The structure elucidation of a natural product requires a set of NMR spectra that includes both carbon observe experiments, such as 1D carbon and DEPT, and proton observe experiments, such as HSQC, HMBC, and COSY. Because NMR probes are optimized for either proton or carbon observe experiments, but not both, this often results in some experiments being acquired at a very suboptimal level of efficiency. An alternative is to deduce the carbon spectrum from the indirect, or f 1, dimension of the heteronuclear 2D experiments. This approach is sometimes being employed for the structure elucidation of newly isolated natural products in cases where the amount of material available precludes carbon observe experiments. However whether this approach is reliable in every case has not yet been established. This study applies the "indirect dimension" approach to a representative set of known natural products. The results are mixed. Analysis of E-HSQC spectra, in conjunction with COSY spectra, reliably defines the carbon spectra of the methyl, methylene, and methine carbons present. However, due to limits in resolution in the f 1 dimension of standard HMBC experiments, the presence and chemical shift positions of some quaternary carbons are fairly frequently obscured by those of other carbons. Thus it is often necessary to acquire a 1D carbon NMR spectrum to support the structure elucidation of a natural product.     

Some phytochemical aspects of medicinal plant research

Various aspects of medicinal plant research, i.e. collection of data, screening for biological activity and isolation and identification of active compounds are briefly discussed with special reference to phytochemical aspects. Based upon this some topics for further methodological research are presented. A strategy for the isolation of pure compounds is outlined. The spectroscopic methods for the identification and structure elucidation of isolated natural products are briefly discussed. The role of each of these methods is a general strategy for product identification and structure elucidation is described.     

天然药物化学史话:“四大光谱”在天然产物结构鉴定中的应用

天然产物化学研究在药物研发中起着非常重要的作用,结构研究又是天然产物化学研究中最重要的工作之一。在天然药物化学史话系列文章的基础上,对在天然产物结构研究中起绝对主导作用的"四大光谱"分析技术,即红外光谱、紫外光谱、质谱、核磁共振波谱在天然产物结构鉴定中的应用历史进行回顾与总结,并对其发展前景进行展望。     

Dolabellanes with antibacterial activity from the brown alga Dilophus spiralis

Seventeen diterpenes featuring the dolabellane skeleton (1-17) were isolated from the organic extracts of the brown alga Dilophus spiralis. Seven compounds are new natural products (1, 3, 5, 6, 11, 14, 15) and eight are structurally revised (2, 4, 7-10, 12, 13), among which three are reported for the first time from a natural source (4, 9, 10). The structure elucidation and the assignment of the relative configurations of the isolated natural products were based on detailed analyses of their spectroscopic data. The structure of metabolite 10 was confirmed by single-crystal X-ray diffraction analysis, whereas the absolute configurations of compounds 2, 4-10, 12, and 13 were determined using the modified Mosher's method on the semisynthetic product 18 and chemical interconversions. The antibacterial activities of compounds 1-18 were evaluated against six strains of Staphylococcus aureus, including multidrug- and methicillin-resistant variants.     

天然药物化学史话:天然产物的生物合成

天然产物因其独特的化学结构,往往具有特定靶点、专一性结合的能力而表现出良好的生物活性,因而一直是生物活性前体化合物和新药发现的重要源泉。植物体通过一次代谢过程和二次代谢过程,仅用简单的原料二氧化碳和水,在酶的作用下就合成了结构千差万别的各类型天然产物。其中一些结构特异、生物活性较强的化合物也成了有机合成化学家们竞相研究的热点。阐明天然产物的生物合成途径,不仅有助于天然产物人工合成的设计和结构推导;同时,生物合成的原理、反应类型及反应机制也为有机合成研究领域提供了灵感,开拓了思路,催生了许多新颖的合成方法;以天然产物化学和分子生物学的发展和融合为基础的化学生物学和合成生物学的诞生将催生下一次生物技术革命。     

Isolation, structure elucidation, and biosynthesis of an unusual hydroxamic acid ester-containing siderophore from Actinosynnema mirum

In this study we report the isolation, structure elucidation, and biosynthesis of mirubactin (1), a siderophore containing an unprecedented chemical functionality in natural products, namely, an O-acyl hydroxamic acid ester. Mirubactin represents the first siderophore isolated from the genus Actinosynnema and the first natural product produced by Actinosynnema mirum whose biosynthetic gene cluster could be identified. Structure elucidation was accomplished through a combination of spectroscopic (NMR, IR, and UV/vis) and mass spectrometric methods and revealed the presence of an unusual ester bond between the δ-N-hydroxyl group of δ-N-formyl-δ-N-hydroxyornithine and a 2,3-dihydroxybenzoate moiety. Bioinformatic analysis of the A. mirum genome and subsequent biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for mirubactin assembly. The proposed biosynthesis of mirubactin comprises the iterative use of a stand-alone carrier-protein-bound substrate, as well as an ester-bond-forming step catalyzed by a C-terminal condensation domain, thus revealing an interesting system for further biochemical studies to gain a deeper understanding of nonribosomal peptide synthetase-catalyzed siderophore biosynthesis.     

Q-TOF及IT-TOF质谱技术在天然产物及其复杂代谢物鉴定中的应用及展望

天然产物在生物体内通常发生广泛代谢,且由于其代谢途径的多样性与复杂性,天然产物代谢物鉴定仍然存在很大的技术挑战。本文综述了复杂基质中代谢物鉴定的质谱技术和分析策略,通过比较目前广泛用于代谢物鉴定的质谱技术的优缺点,以及国内外在代谢物鉴定方面成果,重点讨论和展望了四级杆-飞行时间质谱仪（Q—TOF）、离子阱-飞行时间质谱仪（IT-TOF）技术在天然产物复杂代谢物鉴定方面的巨大优势。根据天然产物的代谢规律,基于质谱技术发展相应的代谢物鉴定策略,Q—TOF与IT-TOF技术的突破和发展将在中药复杂组分及其代谢产物分析领域中发挥重要作用。     

天然药物化学史话:20世纪最伟大的天然有机化学家——Robert Burns Woodward

Robert Burns Woodward是著名的天然有机化学大师。简单介绍Woodward教授生平,回顾他的主要研究成就,尤其在天然产物化学领域的卓越贡献,其中包括天然产物全合成、利用各种光谱手段进行天然产物结构鉴定、化学理论研究、生物合成研究等。谨以此纪念这位20世纪最伟大的有机化学家100周年诞辰。     

新型结构活性天然产物的识别与获取新方法

天然产物与人类健康关系密切。从天然产物中筛选发现新型结构的活性化合物是天然产物化学研究的重点领域。新型结构的活性天然化合物仍是创新药物先导化合物的主要来源之一。天然产物研究者们正在努力寻找有效的新方法，对天然资源样品进行快速分析，在微克级水平高效、快速地鉴定具有结构多样性的天然化合物结构。LC／MS（LC／MS^n）、LC／NMR、LC／NMR／MS、LC／SPE／NMR／MS等色谱-波谱联用技术，在天然产物研究中显示了巨大的威力，是对天然产物进行化学筛选的有效手段，能够达到快速识别和锁定并有针对性地分离获取新型结构化合物的目的。将亲和色谱法与色谱-波谱联用技术结合起来，即将活性筛选与化学筛选在线集成应用，可以大大提高活性天然产物发现的效率和几率，加快药物先导化合物和创新药物的研制速度。     

Bioactive rearranged and halogenated semisynthetic derivatives of the marine natural product sarcophine

Cembranoids are natural diterpenes with 14-membered macrocyclic rings. The simplest natural cembranoid, (+)-cembrene, was isolated from pine oleoresin. Sarcophytols A and B are known cembranoids that inhibit tumor promotion. Sarcophine is a related cembranoid isolated from the Red Sea soft coral Sarcophyton glaucum. Sarcophine and its bioconversion products and semisynthetic derivatives are reported to possess cancer chemopreventive activity. Oxymercuration-demercuration of sarcophine using Hg(OAc)2 and NaBH4 afforded four new rearranged and hydroxylated products. Bromination of sarcophine with N-bromosuccinimide (NBS) furnished two new brominated and rearranged products. Reaction with iodine gave the known iso-sarcophinone and (+)-sarcophytoxin B. Structure elucidation was based on a combination of transition state modeling, molecular dynamics, mechanistic considerations, and 2D NMR data. The antiproliferative activity of the new products is also reported.     

Isolation, structure, and coccidiostat activity of coccidiostatin A

Coccidiosis is one of the more common and costly diseases in poultry that is caused by various Eimeria species. In our quest to discover coccidiostats from natural products, we discovered a microbial fermentation extract that exhibited in vivo anticoccidial activity. Fractionation of this extract led to the discovery of two potent antiprotozoals, emecorrugatin A (1) and coccidiostatin A (2). The former compound exhibited only in vitro activity, whereas the latter new compound exhibited in vivo activity against Eimeria species in chickens at 150 ppm dosed in chicken feed. The isolation, structure elucidation, relative configuration, and activity of coccidiostatin A (2) are described.     

Metabolites of Microcystis aeruginosa bloom material from Lake Kinneret, Israel

Six new metabolites, micropeptin KT1042, microguanidine KT636, aeruginosins KT608A, KT608B, and KT650, and pseudoaeruginosin KT554, were isolated along with the known micropeptins SF909 and HM978, cyanopeptolin S, anabaenopeptin F, and the two isomers of planktocyclin-S-oxide from a bloom material collected from Lake Kinneret, Israel, in March 2005. The structure elucidation and biological activity of the six new natural products isolated from this bloom material and the related aeruginosin GH553 are described.     

Cytotoxic and antibacterial beilschmiedic acids from a Gabonese species of Beilschmiedia

High-throughput natural products chemistry methods have facilitated the isolation of eight new (1-8) and two known (9 and 10) beilschmiedic acid derivatives from the leaves of a Gabonese species of Beilschmiedia. Compounds 3-10 were isolated in microgram quantities, and the NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7 mm MicroCryoProbe. All of the compounds were screened for cytotoxic and antibacterial activity against NCI-H460 human lung cancer cells and a clinical isolate of methicillin-resistant Staphylococcus aureus, respectively. This is the first report of cytotoxic activity for the endiandric/beilschmiedic acid class of compounds.     

Natural products as sources of new drugs over the last 25 years

This review is an updated and expanded version of two prior reviews that were published in this journal in 1997 and 2003. In the case of all approved agents the time frame has been extended to include the 251/2 years from 01/1981 to 06/2006 for all diseases worldwide and from 1950 (earliest so far identified) to 06/2006 for all approved antitumor drugs worldwide. We have continued to utilize our secondary subdivision of a "natural product mimic" or "NM" to join the original primary divisions. From the data presented, the utility of natural products as sources of novel structures, but not necessarily the final drug entity, is still alive and well. Thus, in the area of cancer, over the time frame from around the 1940s to date, of the 155 small molecules, 73% are other than "S" (synthetic), with 47% actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the antiinfective area being dependent on natural products and their structures. Although combinatorial chemistry techniques have succeeded as methods of optimizing structures and have, in fact, been used in the optimization of many recently approved agents, we are able to identify only one de novo combinatorial compound approved as a drug in this 25 plus year time frame. We wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore we consider that this area of natural product research should be expanded significantly.     

Azaphilones from the endophyte Chaetomium globosum

Six new azaphilones, 5'-epichaetoviridin A (7), 4'-epichaetoviridin F (9), 12β-hydroxychaetoviridin C (10), and chaetoviridins G-I (11-13), and six known azaphilones, chaetoviridins A-E (1-5) and 4'-epichaetoviridin A (8), were isolated from the endophytic fungus Chaetomium globosum cultivated in PDB medium for 21 days. The structure elucidation and the assignment of the relative configurations of the new natural products were based on detailed NMR and MS spectroscopic analyses. The structure of compound 1 was confirmed by single-crystal X-ray diffraction analysis. The absolute configurations of compounds 4, 7, 8, and 12 were determined using Mosher's method. The antibiotic activity of the compounds was evaluated using an in vivo Caenorhabditis elegans infection model.     

6-Desmethoxyhormothamnione, a new cytotoxic styrylchromone from the marine cryptophyte Chrysophaeum taylori

Our previous chemical investigations of a yellow marine alga from Puerto Rico, tentatively identified as the tuft-forming cyanobacterium Hormothamnion enteromorphoides, led to the isolation of a structurally novel cytotoxic styrylchromone natural product, hormothamnione [2]. Continued chemical work with this organism has resulted in the isolation and spectroscopic structure elucidation of a closely related styrylchromone, 6-desmethoxyhormothamnione [1], which is also cytotoxic to cancer cells. The substitution pattern of proton, hydroxyl, and methoxyl groups on the chromone ring was established by long range 1H-13C heteronuclear correlation spectroscopy. The taxonomy of the source organism for these styrylchromone natural products, 1 and 2, is revised to the marine cryptophyte Chrysophaeum taylori as a result of detailed microscopic and cultural efforts.     

A phthalide with in vitro growth inhibitory activity from an oidiodendron strain

A screening campaign was implemented utilizing capillary electrophoresis as a primary assay to discover binders to the cancer target Akt1 from a crude natural extract library. Fungal extracts with binding activities were characterized for biochemical inhibition of Akt1 to phosphorylate the downstream substrate protein Bad. One of the crude extracts with bioactivity selected for isolation and structure elucidation from fermentation of the fungal culture Oidiodendron sp. F01895 yielded a new trihydroxy phthalide (1). The structure of 1 was determined by a combination of 1D and 2D NMR spectroscopic data along with high-resolution mass spectrometric data. Compound 1 displays inhibition of Akt1 biochemical activity in vitro and confers growth inhibition on some cancer-derived cell lines in culture.     

Selected topics from forty years of natural products research: betalains to flavonoids, antiviral proteins, and neurotoxic nonprotein amino acids.

The elucidation by NMR and chemical methods of the unique structure of betanidin, the aglycon of the red-violet beet pigment betanin, forty years ago at the University of Zürich, Switzerland, was the beginning of my plant chemistry research program. Many of the same chemical and spectral techniques developed in Zürich have been used at The University of Texas at Austin for the structure analysis of members of many other classes of natural products including especially flavonoids, terpenoids, and alkaloids. Investigations at UT-Austin have concerned many topics such as biochemical and molecular systematics, biosynthetic pathways, structure-activity relationships, and the medicinal importance of natural products and included studies of antiviral proteins in the genus Phytolacca and neurotoxic nonprotein amino acids from cycads and other sources. Following the betalain story and an account of the early development of my UT-Austin biochemical systematic program, the Phytolacca and neurotoxin investigations are discussed herein.     

Countercurrent separation of natural products

An assessment of the technology and method development in countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC), collectively referred to as countercurrent separation (CS), is provided. More than six decades of CS theory and applications are critically reviewed and developed into a practical guide to CS for natural products research. The necessary theoretical foundation is given for better use of CS in the separation of biological molecules of any size, small to large, and from any matrix, simple to complex. The three operational fundamentals of CS--instrumentation, biphasic solvent systems, and theory--are covered in a prismatic fashion. The goal of this review is to provide the necessary background and references for an up-to-date perspective of CS and to point out its potential for the natural products scientist for applications in natural products chemistry, metabolome, and proteome research involving organisms from terrestrial and marine sources.