Microscale methodology for structure elucidation of natural products

Advances in microscale spectroscopic techniques, particularly microcryoprobe NMR, allow discovery and structure elucidation of new molecules down to only a few nanomole. Newer methods for utilizing circular dichroism (CD) have pushed the limits of detection to picomole levels. NMR and CD methods are complementary to the task of elucidation of complete stereostructures of complex natural products. Together, integrated microprobe NMR spectroscopy, microscale degradation and synthesis, are synergistic tools for the discovery of bioactive natural products and have opened new realms for discovery among extreme sources including compounds from uncultured microbes, rare invertebrates and environmental samples.     

基因组挖掘技术在海洋放线菌天然产物研究开发中的应用及展望

利用微生物的基因组信息预测其合成特定天然产物的潜能, 进而进行新化合物分离纯化和结构鉴定的基因组挖掘技术, 已经成为国内外研究的热点, 并在多种细菌和真菌的天然产物发现中得到成功应用。本文综述了基因组挖掘技术的最新进展, 包括生物信息分析和结构预测、基因组指导的天然产物的发现、沉默基因的激活和异源表达技术等, 以及我国学者开发的转录组挖掘技术, 并重点综述了影像质谱技术在基因组挖掘中的应用。目前对海洋放线菌进行基因组挖掘的研究还比较少, 而基因组挖掘技术的发展, 将极大地促进对海洋放线菌天然产物的发现和鉴定。未来除了充分挖掘可培养微生物的基因组, 对未培养微生物宏基因组的挖掘将进一步深入。此外, 除了开发利用基因组中合成天然产物的结构基因和调节基因, 还应该充分开发利用其他不同的遗传元件, 包括不同转录活性和响应不同环境条件和信号的启动子, 以及具有调节作用的RNA等。     

Diversity and biogeography of marine actinobacteria

The actinomycetes, although not all the Actinobacteria, are easy to isolate from the marine environment. However, their ecological role in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate strains for search and discovery of new drugs. However, the marine environment has become a prime resource in search and discovery for novel natural products and biological diversity, and marine actinomycetes turn out to be important contributors. Similarly, striking advances have been made in marine microbial ecology using molecular techniques and metagenomics, and actinobacteria emerge as an often significant, sometimes even dominant, environmental clade. Both approaches - cultivation methods and molecular techniques - are leading to new insights into marine actinobacterial biodiversity and biogeography. Very different views of actinobacterial diversity emerge from these, however, and the true extent and biogeography of this are still not clear. These are important for developing natural product search and discovery strategies, and biogeography is a hot topic for microbial ecologists.     

Natural Products among Brown Algae: The Case of Cystoseira schiffneri Hamel (Sargassaceae,Phaeophyceae)

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone ( 1 ) and a new isololiolide derivative named schiffnerilolide ( 2 ). The structure elucidation and the assignment of relative configurations of the isolated natural products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae belonging to genus Sargassum C. Agardh . Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigment analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes.     

Strategies for dereplication of natural compounds using high-resolution tandem mass spectrometry

Complete structural elucidation of natural products is commonly performed by nuclear magnetic resonance spectroscopy (NMR), but annotating compounds to most likely structures using high-resolution tandem mass spectrometry is a faster and feasible first step. The CASMI contest 2016 (Critical Assessment of Small Molecule Identification) provided spectra of eighteen compounds for the best manual structure identification in the natural products category. High resolution precursor and tandem mass spectra (MS/MS) were available to characterize the compounds. We used the Seven Golden Rules, Sirius2 and MS-FINDER software for determination of molecular formulas, and then we queried the formulas in different natural product databases including DNP, UNPD, ChemSpider and REAXYS to obtain molecular structures. We used different in-silico fragmentation tools including CFM-ID, CSI:FingerID and MS-FINDER to rank these compounds. Additional neutral losses and product ion peaks were manually investigated. This manual and time consuming approach allowed for the correct dereplication of thirteen of the eighteen natural products.     

海绵来源链霉菌S52-B中氨酰胺天然产物的分离与鉴定

【背景】海洋微生物是复杂海洋生态环境中重要的生物资源之一。海洋微生物所产生的活性天然产物极为丰富,是药物或药物先导化合物的重要来源。【目的】探索海洋中海绵来源链霉菌Streptomycessp.S52-B的优势生长条件,挖掘其次级代谢产物,以期分离具有良好生物活性的天然产物。【方法】根据"One Strain Many Compounds"(OSMAC)策略,寻找利于Streptomyces sp. S52-B生长和次级代谢产物产生的优势培养基,结合质谱及特征性的紫外吸收谱图,选择培养基进行大量发酵。利用正相硅胶柱色谱、葡聚糖凝胶柱色谱和制备型高效液相色谱等进行分离纯化,并应用高分辨质谱和核磁共振光谱进行化合物结构解析。【结果】确定培养基A–D为海洋链霉菌S52-B的优势培养基,基于紫外吸收光谱与质谱分析,从培养基A的大量发酵物中分离鉴定3个具有吡咯并[4,3,2-de]喹啉核心结构的含氯化合物,属于氨酰胺类天然产物,其中Ammosalic acid为新结构化合物。【结论】已知含有吡咯并喹啉母核的氨酰胺类家族化合物具有优良的抗癌活性。本研究从海绵来源链霉菌S52-B中分离鉴定了3个氨酰胺类化合物,其中一个是新结构化合物,不仅丰富了此类化合物家族的结构类型,也为研究其生物合成途径中的未知机理奠定了基础,还有利于结合培养条件和基因组信息从这株海绵来源链霉菌中挖掘新结构的活性天然产物。     

Drug Discovery from Marine Microbes

The marine environment has been a source of more than 20,000 inspirational natural products discovered over the past 50 years. From these efforts, 9 approved drugs and 12 current clinical trial agents have been discovered, either as natural products or as molecules inspired from the natural product structure. To a significant degree, these have come from collections of marine invertebrates largely obtained from shallow-water tropical ecosystems. However, there is a growing recognition that marine invertebrates are oftentimes populated with enormous quantities of “associated” or symbiotic microorganisms and that microorganisms are the true metabolic sources of these most valuable of marine natural products. Also, because of the inherently multidisciplinary nature of this field, a high degree of innovation is characteristic of marine natural product drug discovery efforts.     

Cocon: From NMR Correlation Data to Molecular Constitutions

The constitutional assignment of natural products by NMR spectroscopy is usually based on 2D NMR experiments like COSY, HSQC, and HMBC. In this paper, the resulting connectivity information is used as input for the new structure generating program Cocon which both improves and dramatically accelerates the process of constitutional assignment. Cocon allows to quantify the value of connectivity information (2D NMR correlation data) for structure elucidation problems. Applying Cocon, it is systematically evaluated to which degree the NMR experiments COSY, ¹H,¹³C-HMBC and 1,1-ADEQUATE constrain the number of constitutions compatible with the data sets of two secondary metabolites from marine sponges.Electronic Supplementary Material available.     

Design of compound libraries based on natural product scaffolds and protein structure similarity clustering (PSSC)

Recent advances in structural biology, bioinformatics and combinatorial chemistry have significantly impacted the discovery of small molecules that modulate protein functions. Natural products which have evolved to bind to proteins may serve as biologically validated starting points for the design of focused libraries that might provide protein ligands with enhanced quality and probability. The combined application of natural product derived scaffolds with a new approach that clusters proteins according to structural similarity of their ligand sensing cores provides a new principle for the design and synthesis of such libraries. This article discusses recent advances in the synthesis of natural product inspired compound collections and the application of protein structure similarity clustering for the development of such libraries.     

An NMR method towards the routine chiral determination of natural products

State-of-the-art structure elucidation and dereplication of natural products is incomplete without the determination of enantiomeric purity, especially when compounds are to be biologically evaluated. An NMR procedure is presented in order to distinguish and determine enantiomers in natural product samples. The method is also of value in the structure elucidation process by providing information, which is otherwise of a non-routine nature. Using enantiomeric 1-acetoxychavicol acetates and carvones as model compounds, this study presents a chiral NMR procedure that allows distinction and determination of chiral antipodes of natural products in a routine set-up.     

Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates

In the past few decades, marine natural products bioprospecting has yielded a considerable number of drug candidates. Two marine natural products have recently been admitted as new drugs: Prialt (also known as ziconotide) as a potent analgesic for severe chronic pain and Yondelis (known also as trabectedin or E-743) as antitumor agent for the treatment of advanced soft tissue sarcoma. In this protocol, methods for bioactivity-guided isolation, purification and identification of secondary metabolites from marine invertebrates such as sponges, tunicates, soft corals and crinoids are discussed. To achieve this goal, solvent extraction of usually freeze-dried sample of marine organisms is performed. Next, the extract obtained is fractionated by liquid-liquid partitioning followed by various chromatographic separation techniques including thin layer chromatography, vacuum liquid chromatography, column chromatography (CC) and preparative high-performance reversed-phase liquid chromatography. Isolation of bioactive secondary metabolites is usually monitored by bioactivity assays, e.g., antioxidant (2,2-diphenyl-1-picryl hydrazyl) and cytotoxicity (microculture tetrazolium) activities that ultimately yield the active principles. Special care should be taken when performing isolation procedures adapted to the physical and chemical characteristics of the compounds isolated, particularly their lipo- or hydrophilic characters. Examples of isolation of compounds of different polarities from extracts of various marine invertebrates will be presented in this protocol. Structure elucidation is achieved using recent spectroscopic techniques, especially 2D NMR and mass spectrometry analysis.     

Renewed interests in the discovery of bioactive actinomycete metabolites driven by emerging technologies

Actinomycetes are prolific sources of bioactive molecules. Traditional workflows including bacterial isolation, fermentation, metabolite identification and structure elucidation have resulted in high rates of natural product rediscovery in recent years. Recent advancements in multi-omics techniques have uncovered cryptic gene clusters within the genomes of actinomycetes, potentially introducing vast resources for the investigation of bioactive molecules. While developments in culture techniques have allowed for the fermentation of difficult-to-culture actinomycetes, high-throughput metabolite screening has offered plenary tools to accelerate hits discovery. A variety of new bioactive molecules have been isolated from actinomycetes of unique environmental origins, such as endophytic and symbiotic actinomycetes. Synthetic biology and genome mining have also emerged as new frontiers for the discovery of bioactive molecules. This review covers the highlights of recent developments in actinomycete-derived natural product drug discovery.     

Cultivation-independent approaches to investigate the chemistry of marine symbiotic bacteria

Sessile marine animals like sponges, tunicates, and bryozoans are a rich source of bioactive natural products, many of which exhibit potent anticancer activities. However, most of these substances are available in very limited amounts only, which has prohibited further drug development. Recent evidence suggests that symbiotic bacteria might be the true producers of many animal-derived metabolites. In addition to revealing fascinating perspectives for research in marine chemical ecology, these findings suggest new solutions to the supply problem. Although most symbionts remain uncultivated, bacterial production systems might be created by isolating biosynthetic genes from marine metagenomes, and expressing them in culturable bacterial hosts. This review discusses cell-sorting, natural product visualization, and phylogenetic approaches to identify symbiotic producers. In addition, strategies to isolate genes and gene clusters from marine species consortia are described. These techniques have provided insights into the bacterial origin and biosynthesis of polyketides like the onnamides, swinholides, and bryostatins, of peptides including the patellamides, chlorinated dipeptides, and theopalauamide as well as of brominated biphenylethers.     

Engineering natural products using combinatorial biosynthesis and biocatalysis

Many biologically active natural products are produced by the host organisms using dedicated biosynthetic pathways. The programming rules of these pathways may be rationally manipulated through combinatorial biosynthesis to produce natural products that contain structural variations or enhanced pharmacological properties. Furthermore, these pathways contain enzymes that can be harvested as powerful biocatalysts for the synthesis of both new drugs and existing blockbuster therapeutics. This review will highlight recent advances in exploring natural product biosynthetic pathways for new compounds, novel enzymes and useful biocatalysts.     

Transformations of morphine alkaloids by Pseudomonas putida M10.

The oxidation of morphine by washed-cell incubations of Pseudomonas putida M10 gave rise to a large number of transformation products including hydromorphone (dihydromorphinone), 14 beta-hydroxymorphine, 14 beta-hydroxymorphinone, and dihydromorphine. Similarly, in incubations with oxymorphone (14 beta-hydroxydihydromorphinone) as substrate, the major transformation product was identified as oxymorphol (14 beta-hydroxydihydromorphine). The identities of all these biological products were confirmed by mass spectrometry and 1H nuclear magnetic resonance spectroscopy. This is the first report describing structural evidence for the biological synthesis of 14 beta-hydroxymorphine and 14 beta-hydroxymorphinone. These products have applications as intermediates in the synthesis of semisynthetic opiate drugs.     

Indole alkaloid marine natural products: an established source of cancer drug leads with considerable promise for the control of parasitic, neurological and other diseases

The marine environment produces natural products from a variety of structural classes exhibiting activity against numerous disease targets. Historically marine natural products have largely been explored as anticancer agents. The indole alkaloids are a class of marine natural products that show unique promise in the development of new drug leads. This report reviews the literature on indole alkaloids of marine origin and also highlights our own research. Specific biological activities of indole alkaloids presented here include: cytotoxicity, antiviral, antiparasitic, anti-inflammatory, serotonin antagonism, Ca-releasing, calmodulin antagonism, and other pharmacological activities.     

Bioactive Pimarane‐Type Diterpenes from Marine Organisms

Marine organisms represent an abundant sources of bioactive natural products with unique chemical structure. Pimarane diterpenes are a structurally diverse family of natural products with various biological activities and pharmacological properties. A prolific source of new marine‐derived pimarane diterpenes have attracted considerable interest because of their unique structural diversity and varied biological activities, including antimicrobial, antitumor, and antiproliferative activities. This review provides a comprehensive overview of the structures, names, bioactivities, and references of 80 marine‐derived pimarane‐type natural products for the first time.     

Antifouling activity of synthetic γ-hydroxybutenolides

The desire to provide greener antifouling (AF) biocides for incorporation into marine paints has led to the discovery of many AF natural products. However, economical and sustainable supply of these natural products is often lacking. Twenty one structurally diverse γ-hydroxybutenolides – inspired by two natural AF sesterterpene γ-hydroxybutenolides (cavernosolide and lintenolide A) – were synthesized and tested for in vitro AF efficacy against the marine bryozoan larvae (Bugula neritina) and algae (Isochrysis galbana). The most potent analogues (EC₅₀ ca. 2 μg/mL) had AF activity comparable to the natural products (EC₅₀ ca. 0.6 μg/mL). Sites of attachment of side chains to the γ-hydroxybutenolide core structure had little effect on AF activity, however a clear relationship was established between lipophilicity and AF activity. Four analogues were chosen for field trials, incorporated into paints and deployed on panels in the marine environment. These analogues were chosen to reflect logistics of large scale synthesis and to provide representation of diverse structural motifs. Without any primary or co-biocides (except ZnO as a paint pigment), two analogues demonstrated a pronounced AF effect by resisting biofouling for over 18 weeks, compared to the ZnO control of between 7 and 10 weeks. Fine tuning of the structural motifs and/or the inclusion of co-biocides in paint formulations provide the potential to increase AF efficacy further.