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。     

Isolation and structure determination of nostocyclopeptides A1 and A2 from the terrestrial cyanobacterium Nostoc sp. ATCC53789

The isolation and total structure determination of nostocyclopeptides A1 (1) and A2 (2) are described. These cyclic heptapeptides, which possess a unique imino linkage in the macrocyclic ring, are characteristic constituents of the cryptophycin-producing cyanobacterium Nostoc sp. ATCC53789. 1D TOCSY experiments proved to be very useful in identifying the seven amino acid residues in each compound, and HMBC and NOESY correlations made it possible to sequence the seven units into a total gross structure. The absolute stereochemistry was determined by directly comparing the amino acids in the acid hydrolyzate of each natural product and its peroxide oxidation and borohydride reduction products with authentic standards. Studies were carried out on the biosynthesis and initiated on the biological activity of these cyclic peptides.     

Genetic evidence supports secondary metabolic diversity in Prochloron spp., the cyanobacterial symbiont of a tropical ascidian

Didemnid family ascidians commonly harbor obligate cyanobacterial symbionts, Prochloron spp., which have been proposed to biosynthesize cyclic peptides. Here, it is shown that Prochloron spp. do indeed contain genes for nonribosomal peptide biosynthesis, although genes for cyclic peptide biosynthesis have not yet been characterized. A peptide synthetase-containing open reading frame of unknown function was cloned from the Prochloron symbionts of some didemnid ascidians, but not from others. These data indicate that Prochloron spp. have variable secondary metabolic potential.     

Synthesis of the marine sponge cycloheptapeptide phakellistatin 5(1)

Phakellistatin 5 (1), a constituent of The Federated States of Micronesia (Chuuk) marine sponge Phakellia costada, was synthesized by solution-phase and solid-phase techniques. Because the linear peptide bearing (R)-Asn resisted cyclization, the synthesis of this peptide was repeated using the PAL resin attachment proceeding from N-Fmoc-D-Asp-alpha-OCH(2)CH=CH(2). After addition of the final unit (Ala), the allyl ester was removed under neutral conditions with Pd(o) [P(C(6)H(5))(3)](4). Removal of the final Fmoc-protecting group and cyclization with PyAOP provided (R)-Asn-phakellistatin 5 (2) in 28% overall yield. The same synthetic route from (S)-Asp led to natural phakellistatin 5 (1) in 15% overall recovery. The solution-phase and solid-phase synthetic products derived from (S)-Asp were found to be chemically but not biologically identical with natural phakellistatin 5 (1). This important fact suggested that a trace, albeit highly cancer-cell growth inhibitory, constituent accompanied the natural product or that there is a subtle conformational difference between the synthetic and natural cyclic peptides.     

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。     

Gene mining and efficient biosynthesis of a fungal peptidyl alkaloid

Objective: Peptidyl alkaloids, a series of important natural products can be assembled by fungal nonribosomal peptide synthetases(NRPSs). However, many of the NRPSs associated gene clusters are silent under laboratory conditions, and the traditional chemical separation yields are low. In this study, we aim to discovery and efficiently prepare fungal peptidyl alkaloids assembled by fungal NRPSs.Methods: Bioinformatics analysis of gene cluster containing NRPSs from the genome of Penicillium thymicola, and heterologous expression of the putative gene cluster in Aspergillus nidulans were performed.Isolation, structural identification, and biological evaluation of the product from heterologous expression were carried out.Results: The putative tri-modular NRPS Anc A was heterologous-expressed in A. nidulans to give anacine(1) with high yield, which showed moderate and selective cytotoxic activity against A549 cell line.Conclusion: Heterologous expression in A. nidulans is an efficient strategy for mining fungal peptidyl alkaloids.     

基于液质联用数据库技术的中药及天然产物化学成分快速鉴定方法的建立

目的:应用液质联用数据库技术建立中药及天然产物化学成分的快速鉴定方法。方法:采用超高效液相色谱电喷雾离子化四级杆飞行时间串联质谱(UPLC-ESI-Q-TOF)技术,采集不同类型中药及天然产物化学成分的保留时间和质谱信息并建立质谱信息库,应用Masslynx工作站的Chromalynx XS模块的Identify功能,自动快速鉴定色谱图中各个色谱峰;运用保留时间校正方法,对相同化合物在不同色谱系统下的保留时间进行校正,以扩大该方法适应性。结果:该方法中建立的质谱信息库现收录了包含生物碱类、黄酮类、醌类、苯丙素类、萜类等不同类型的210余个化学成分的液质联用信息,包括正负离子模式下各成分的保留时间,准分子离子峰及其碎片离子的精确质量数。使用该方法进行样品成分鉴定测试,检出准确率90%左右。结论:与人工成分鉴定相比较,该方法简便、快捷、准确率高。保留时间校正方法的应用,一定程度上解决了不同色谱系统下保留时间不同导致质谱库无法统一的问题,扩大了本方法的应用范围。     

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??Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are two kinds of multi-modular enzymes, which biosynthesize highly complex polyketides and nonribosonmal peptides, respectively. Both of these two secondary metabolites are of considerable pharmaceutical relevance and are thought to cover diverse biological functions. With the development of sequencing and bioinformatics, data about PKS/NRPS are increasing rapidly. New PKS/NRPS databases are created to analyze gene sequence and predict the functions and structures of natural products. In this article, we introduce five newest databases including PKMiner, NRPSsp, NaPDoS, ClusterMine360, and IMG-ABC, with the goal to help researchers choose databases.     

海绵药物的研究进展:化学和生物活性

近年来国际上对海洋天然产物的研究日益深入，依托现代大规模药理筛选，目前已有多种结构新颖、药理活性显著且作用机制特殊的海洋天然产物进入临床试验阶段或正在进行临床前研究。海绵种类繁多，代谢途径独特，生存环境多样，共生现象复杂而普遍，多年来一直是海洋天然产物领域最富成果的研究领域之一。本文简要介绍近几年海绵化学成份及其药理活性的研究进展，以供国内同行参考。     

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??OBJECTIVE To identify the metabolites of imperatorin in rat urine, feces and bile after oral administration as well as the transformation products of imperatorin after incubation with rat liver microsomes with HPLC-QTrap-MS technology. METHODS The combination use of HPLC-QTrap-MS scanning mode including multiple ion monitoring-information dependent acquisition-enhanced product ion (MRM/MIM-IDA-EPI)mode, precursor scan-enhanced resolution-information dependent acquisition-enhanced product ion (PREC-ER-IDA-EPI)mode and enhanced product ion (EPI) mode were performed for the identification of the metabolites. Based on the simultaneous appearance of [M+H]⁺ and [M+NH₄]⁺ in the spectrum of PREC, the molecular weight could be unambiguously identified. The structures of compounds were then identified by the fragment ions generated from these three modes. RESULTS With the HPLC-QTrap-MS method, 32 metabolites in urine sample, 14 metabolites in faces sample, 6 metabolites in bile sample and 17 transformation products from the rat liver microsomes sample were detected. CONCLUSION Imperatorin is metabolized mainly in liver and excreted through kidney. The metabolic profiles of imperatorin in vivo and in vitro have good correlation.     

Psychrophilin A and cycloaspeptide D, novel cyclic peptides from the psychrotolerant fungus Penicillium ribeum

Two fungal metabolites, psychrophilin A (1) and cycloaspeptide D (2), together with the known cycloaspeptide A (3) were isolated from the psychrotolerant fungus Penicillium ribeum using high-speed countercurrent chromatography (HSCCC) and preparative HPLC. The structures were determined from 1D and 2D NMR techniques, HREIMS, tandem mass spectrometry (ESMS/MS), and X-ray crystallography. The amino acid residues of psychrophilin A (1) and cycloaspeptide D (2) were all found to possess the l configuration by Marfey's method. Psychrophilin A (1) is the first natural cyclic peptide containing a nitro group instead of an amino group.     

Purity-activity relationships of natural products: the case of anti-TB active ursolic acid

The present study explores the variability of biological responses from the perspective of sample purity and introduces the concept of purity-activity relationships (PARs) in natural product research. The abundant plant triterpene ursolic acid (1) was selected as an exemplary natural product due to the overwhelming number yet inconsistent nature of its approximate 120 reported biological activities, which include anti-TB potential. Nine different samples of ursolic acid with purity certifications were obtained, and their purity was independently assessed by means of quantitative 1H NMR (qHNMR). Biological evaluation consisted of determining MICs against two strains of virulent Mycobacterium tuberculosis and IC50 values in Vero cells. Ab initio structure elucidation provided unequivocal structural confirmation and included an extensive 1H NMR spin system analysis, determination of nearly all J couplings and the complete NOE pattern, and led to the revision of earlier reports. As a net result, a sigmoid PAR profile of 1 was obtained, demonstrating the inverse correlation of purity and anti-TB bioactivity. The results imply that synergistic effects of 1 and its varying impurities are the likely cause of previously reported antimycobacterial potential. Generating PARs is a powerful extension of the routinely performed quantitative correlation of structure and activity ([Q]SAR). Advanced by the use of primary analytical methods such as qHNMR, PARs enable the elucidation of cases like 1 when increasing purity voids biological activity. This underlines the potential of PARs as a tool in drug discovery and synergy research and accentuates the need to routinely combine biological testing with purity assessment.     

慢性肾衰尿液样本代谢组学研究

研究基于气-质联用(GC-MS)技术的尿液代谢组学预处理方法,对慢性肾功能衰竭患者与正常对照组人群尿液样本进行代谢组学研究。收集慢性肾功能衰竭患者尿液样本54例;正常对照组尿液样本25例。对所有样本采用以葵酸作为内标的硅烷化衍生反应,及GC-MS分析测试方法得到谱图之后,通过层次聚类分析和主成份分析及ROC曲线比较慢性肾功能衰竭与正常对照组之间谱峰的差异性,并利用NIST谱库、Mainlib数据库和Wiley数据库鉴定差异性代谢产物。慢性肾衰患者和正常人的尿液样本,共筛选出有显著性差异(P0.05)的峰有8个。其代谢物为:肌醇、L(-)-阿卓糖、D-呋喃木糖、己糖醇、核糖酸、2-甲氧羰基-3-甲基-3-丁烯酸甲酯、丙酸、廿二烷。此结果显示气-质联用(GC-MS)技术能为慢性肾衰的物质基础进行标志物的筛选,为慢性肾功能衰竭诊断研究提供一定的科研思路与方法。     

超高效液相色谱-质谱联用技术评价当归养血丸中阿胶投料情况

目的 建立当归养血丸中阿胶的检测方法,为投料阿胶质量控制提供技术支持。方法 采用胰蛋白酶对当归养血丸进行酶解,利用超高效液相色谱-质谱联用技术(UPLC-MS/MS),在电喷雾离子化(ESI⁺)模式下,进行多反应监测(multiple reaction monitoring,MRM),进行阿胶专属鉴别、马皮源成分检查及阿胶特征多肽含量测定。结果 建立方法线性、专属性、稳定性、重复性、耐用性良好。以此方法,11批当归养血丸中均检出阿胶,其中9批检出马皮源成分,7批低于阿胶特征多肽含量拟定限度。结论 所建立的方法准确可靠、专属性强,可用于当归养血丸中阿胶的质量评价。     

Do plant cyclotides have potential as immunosuppressant peptides?

Cyclotides are an abundant and diverse group of ribosomally synthesized plant peptides containing a cyclic cystine-knotted structure that confers them with remarkable stability. They are explored for their distribution in plants, although little is known about the individual peptide content of a single species. Therefore, we chemically analyzed the crude extract of the coffee-family plant Oldenlandia affinis using a rapid peptidomics workflow utilizing nano-LC-MS, peptide reconstruct with database identification, and MS/MS automated sequence analysis to determine its cyclotide content. Biologically, cyclotides are mainly explored for applications in agriculture and drug design; here we report their growth-inhibiting effects on primary cells of the human immune system using biological and immunological end points in cell-based test systems. LC-MS quantification of the active O. affinis plant extract triggered the characterization of the antiproliferative activity of kalata B1, one of the most abundant cyclotides in this extract, on primary activated human lymphocytes. The effect has a defined concentration range and was not due to cytotoxicity, thus opening a new avenue to utilize native and synthetically optimized plant cyclotides for applications in immune-related disorders and as immunosuppressant peptides.     

具有降血糖活性的生物碱及其作用机制

生物碱是天然有机体中具有众多结构类型和显著生物活性的一类次级代谢产物,大多具有复杂的环状结构,具有降血糖、抗肿瘤、抗菌、抗病毒等药理活性,是药用植物中最重要的活性成分之一。针对国内外研究的降血糖活性生物碱及其作用机制进行综述,以期为生物碱类降血糖药物的发现、药理学、构效关系等研究提供参考依据。     

Dereplication, residual complexity, and rational naming: the case of the Actaea triterpenes

The genus Actaea (including Cimicifuga) has been the source of ～200 cycloartane triterpenes. While they are major bioactive constituents of complementary and alternative medicines, their structural similarity is a major dereplication problem. Moreover, their trivial names seldom indicate the actual structure. This project develops two new tools for Actaea triterpenes that enable rapid dereplication of more than 170 known triterpenes and facilitates elucidation of new compounds. A predictive computational model based on classification binary trees (CBTs) allows in silico determination of the aglycone type. This tool utilizes the Me (1)H NMR chemical shifts and has potential to be applicable to other natural products. Actaea triterpene dereplication is supported by a new systematic naming scheme. A combination of CBTs, (1)H NMR deconvolution, characteristic (1)H NMR signals, and quantitative (1)H NMR (qHNMR) led to the unambiguous identification of minor constituents in residually complex triterpene samples. Utilizing a 1.7 mm cryo-microprobe at 700 MHz, qHNMR enabled characterization of residual complexity at the 10-20 μg level in a 1-5 mg sample. The identification of five co-occurring minor constituents, belonging to four different triterpene skeleton types, in a repeatedly purified natural product emphasizes the critical need for the evaluation of residual complexity of reference materials, especially when used for biological assessment.     

天然药物化学史话:来自海洋的药物

海洋独特的环境造就了海洋生物的多样性和海洋生物次生代谢产物的多样性,海洋天然产物新颖的化学结构和独特的生物活性成为新药研发先导化合物的宝库。主要针对近年来已经上市或者具有良好开发前景的海洋药物的发现过程及化学结构进行简要介绍,以期能引起更多专业工作者的兴趣,并借此激励有志于科学研究的年轻工作者。     

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.     

The guineamides,novel cyclic depsipeptides from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula

The guineamides (1-6) are novel cyclic depsipeptides isolated and characterized from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula. The planar structures of these new natural products were established using an extensive array of 1D and 2D NMR experiments, including HSQC, TOCSY, and HMBC. Absolute stereochemistry was determined using a combination of chemical manipulations as well as Marfey's method. These metabolites all contain beta-amino or beta-hydroxy carboxylic acid residues, an increasingly common feature in marine cyanobacterial metabolites. The identification of 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha) in guineamides E (5) and F (6) represents the first report of such a residue in a natural product. In addition, characterization of the unique beta-amino acid 2-methyl-3-aminopentanoic acid (Mapa) in guineamide A (1), which has also been reported as a component of several marine molluscan metabolites, especially from those of Dolabella auricularia, further supports the diet-derived nature of such compounds as isolated from marine invertebrates. Guineamides B (2) and C (3) possess moderate cytotoxicity to a mouse neuroblastoma cell line with IC(50) values of 15 and 16 microM, respectively.