摘要
由于其自身特殊的生存环境，海洋来源放线菌具有复杂独特的代谢途径，产生了许多结构新颖、生物活性良好的次级代谢产物，这些活性代谢产物为新药的开发提供了丰富的先导化合物，有些已经进入临床前研究。为开发海洋来源放线菌资源，探寻新结构的生物活性化合物，本文选取了海洋生境来源的五株放线菌作为目标菌株，进行次级代谢产物的化学结构和生物活性研究。研究的主要内容包括以下几部分：1）海洋来源放线菌分离方法的摸索与菌株的分离；2）菌株的化学筛选、活性筛选与分子探针筛选；3）次级代谢产物的分离纯化；4）单体化合物平面结构和立体结构的确定；5）单体化合物生物活性的初步评价。
综合运用海洋放线菌分离在样品预处理，选择性培养基配制，抑制剂选择方面的部分方法，从十二个海洋来源的样品中分离得到放线菌136株。对实验室288株海洋来源的放线菌进行了化学筛选，经过初步排重与再排重得到103株放线菌，丰富了实验室的放线菌TLC和HPLC指纹图谱库。对103株放线菌进行了P388肿瘤细胞的活性筛选，得到抗肿瘤活性菌株25株，为海洋抗肿瘤活性先导化合物的研究提供了活性菌株资源。设计了基于PKS、NRPS、PT和卤代酶等功能基因的特异性引物对菌株进行基因筛选，结合活性和化学筛选，选取了一株分子探针筛选呈阳性、具有抗肿瘤活性和丰富TLC薄层行为和HPLC图谱特征的放线菌CHQ-64为本文的重点实验菌株。本论文另外4株实验菌株，包括3株海泥或海绵来源的高活性抗肿瘤菌株，以及1株海绵来源的具有丰富的TLC薄层行为和特殊的HPLC图谱特征的菌株。
对目标菌株发酵产物通过各种常规柱色谱及高效液相色谱等化学分离纯化手段，从中国南海红树林海泥来源放线菌CHQ-64的放线菌2号培养基摇床培养提取物中分离得到42个化合物(1-42)，从其放线菌2号培养基静置培养提取物中分离得到7个化合物(43-49)；从广东硇洲岛海绵样品来源放线菌CMN-62的代谢产物中分离得到7个化合物(50-56)；从西沙海绵样品来源放线菌CMN-1的代谢产物中分离得到8个化合物(57-64)；从胶州湾海泥样品来源放线菌CHQ-2的代谢产物中分离得到3个化合物(65-67)；从胶州湾海泥样品来源放线菌CHQ-25的代谢产物中分离得到3个化合物(68-70)，共70个化合物。
利用理化性质和波谱学方法(MS, IR, UV, CD, NMR, CD量子计算and X-ray)阐明了70个化合物的化学结构(Fig.1)。化合物的结构类型包括吲哚萜类化合物7个(1-6,43)，多烯大环内酯类化合物6个(7-12)，生物碱类化合物28个{包括吡咯类化合物6个(13-18)，杀粉蝶菌素类化合物5个(43-47)，吲哚咔唑类化合物3个(68-70)，其它生物碱类化合物14个(25-32,37,39-41,55-56)}，聚酮长链类化合物2个(48-49)，环二肽类化合物9个(19-24,60-62)，苯衍生物5个(33-36,54)，氨基糖苷类衍生物4个(50-53)，γ-丁内酯衍生物3个(57-59)，放线菌素类化合物3个(65-67)和其它类化合物3个(38,63-64)。其中发现新化合物23个，包括2个具有罕见稠环形式的吲哚萜类化合物(1-2)，4个结构新颖的drimentine类化合物(3-5,43)，6个多烯大环内酯类化合物(7-12)，3个生物碱类化合物(14-15,40)，2个苯衍生物(35-36)，2个聚酮长链类化合物(48-49)，4个氨基糖苷类衍生物(50-53)。采用NOESY、X-Ray、CD以及CD量子计算等方法确定了吲哚萜类化合物(1-5)的相对及绝对构型；采用生合成关系、偶合常数对比、Mosher反应等方法确定了多烯大环内酯类化合物(7-12)和氨基糖苷类衍生物(50-53)的相对及绝对构型。
在活性方面，对于新骨架indotertine类化合物，含醛基的化合物2显示了对HCT-8和A-549两种肿瘤细胞的抑制活性，IC50分别是6.96和4.88μM;属于drimentine类化合物的新化合物4，显示了对HCT-8，Bel-7402，A549和A2780四种肿瘤细胞的抑制活性, IC50分别是2.81，1.38，1.01和2.54μM，显示了很好的抗肿瘤活性，已经申请专利。采用96孔板的方法，以制霉菌素为阳性药，评测了6个多烯大环内酯类化合物对白色念珠菌的抑制活性，其中化合物7有较强的抗Candida albicans活性，MICs是25-50μM，与阳性药制霉菌素相当。CMN-1中分离得到的3个γ-丁内酯衍生物(57-59)均显示具有群体感应活性，其中化合物59有较强的群体感应活性。部分化合物的生物活性评价仍在进行中。
综上所述，本文共阐明了5株海洋来源放线菌中70个化学成分的结构，其中包括新化合物23个。在新化合物的研究中，本文首次发现由色氨酸和倍半萜杂合途径生成的五元环骈合形式的吲哚倍半萜类化合物(1-2)；从天然界得到具有呋喃环的结构新颖的多烯大环内酯类化合物(12)；从微生物中得到的罕见的氨基糖苷类衍生物(53)。在活性研究中，发现了2个抗肿瘤活性较强的新化合物(2,4)；1个抗真菌活性较强的新化合物(7)；首次发现已知化合物59具有显著的群体感应活性。上述研究为海洋天然产物化学提供了结构新颖的化合物骨架，为抗肿瘤、抗菌等生物活性研究提供了活性化合物模板。本文的重点实验菌株CHQ-64，PKSI和NRPS分子探针筛选呈阳性、具有抗肿瘤活性和丰富TLC薄层行为和HPLC图谱特征，从中分离得到49个化合物，新化合物19个，涵盖多种化合物结构类型，实验结果表明，基因-活性-化学集成筛选的方法，可以在一定程度上能够克服放线菌株重复研究和单纯以生物活性为导向的天然产物分离中高活性已知化合物出现几率高的问题。这一研究结果，也为今后深入开展该领域的研究提供了一定的实验基础。
Due to the special living environment, marine-derived actinomycetes possesscomplex and distinct metabolic capabilities, resulting in wide diversity of theirsecondary metabolites in chemical structure and biological activity. Among them,many valuable leading compounds were obtained for discovery of new antibiotics,and some had been selected for preclinical study. In order to search for metaboliteswith chemical novelty and outstanding biological activities, a study on marine-derivedactinomycetes was carried out. Studies include isolating and screening strains,purification and structural elucidation of the secondary metabolites, and preliminarybioactivities evaluation of some pure compounds.
Comprehensive methods including sample pretreatment, selective medium,inhibitor selection were used and136actinomycetes were isolated from twevelmarine-derived soil samples. Applying chemical screen assays,103actinomyceteswere selected from288actinomycetes of our laboratory, which has enriched our TLCand HPLC fingerprint library. Using the bioactive screen,25antitumor strains wereobtained from the103actinomycetes. Actinomycetes CHQ-64, which was positive toboth PKS I and NRPS gene fragments, having anti-tumor activity and rich TLCthinlayer behavior and HPLC chromatogram characteristics, was selected as a mainexperimental strain by PKS I/NRPS/helogenase gene fragments assays, together withapplying activity and chemical screening. The strains CMN-1/CHQ-2/CHQ-25wereselected because of significant antitumor activity and Streptomyces sp. CMN-62wasfocus on its rich TLC thinlayer behavior and HPLC chromatogram characteristics.
In all,70compounds were separated and purified, from the extracts of the5aimed strains, by means of solvent extraction, silica gel column, ODS-C18, SephadexLH20, PHPLC, and etc. From Streptomyces sp. CHQ-64,49compounds (1-49) were isolated; from Streptomyces sp. CMN-62,7compounds (50-56) were isolated; fromStreptomyces sp. CMN-1,8compounds (57-64) were isolated; from Streptomyces sp.CHQ-2,3compounds (65-67) were isolated; from Streptomyces sp. CHQ-25,4compounds (68-70) were isolated.
Structures of the70compounds were respectively determined by theirphysico-chemical properties and spectral data (MS, IR, UV, CD, NMR, TDDFT ECDcalculations and X-ray). Among them there are7hybrid isoprenoids (1-6,43),6oxopolyenes (7-12),28alkaloids {including6pyrrole derivatives (13-18),5piericidinderivatives (43-47),3indolecarbazole derivatives (68-70) and other14alkaloids(25-32,37,39-41,55-56)},2polyketides (48-49),9cycloheximide-derivates (19-24,60-62),5benzol derivatives (33-36,54),4aminobenzoic acid glycosides derivatives(50-53),3γ-butyrolactone derivatives (57-59),3actinomycins (65-67) and otherstructural type compounds (38,63-64). Twenty-five compounds are new, including2hybrid isoprenoids (1-2) with a rare form of fused rings,4novel hybrid isoprenoids(3-5,43),6oxo polyenes (7-12),3alkaloids (14-15,40),2benzol derivatives (35-36),2polyketides (48-49),4aminobenzoic acid glycosides derivatives (50-53). In thestudy, we established the relative and absolute configurations of hybrid isoprenoids(1-5) by the combination of NOESY, X-Ray, CD, and TDDFT ECD calculations. Therelative and absolute configurations of oxo polyenes (7-12) and aminobenzoic acidglycosides (50-53) were determined by the combination of biosynthetic origin,conformational analysis, coupling constants, and mosher methods.
In the bioactivities evaluation, with respect to the new skeleton class, compound2has a formyl group which showed the best cytotoxic activities against HCT-8andA549cell lines, with IC50values of6.96and4.88μM, respectively. For drimentines,compound4showed the best cytotoxic activities against HCT-8, Bel-7402, A549andA2780cell lines, with IC50values of2.81,1.38,1.01and2.54μM, respectively. Apatent application for drimentine G (4) had been filed for in China. The biologicalactivities of six polyene macrolides were evaluated by antifungal assay with Candidaalbicans, using nystatin as a positive control. Compound7showed pronounced antifungal activity against Candida albicans with MICs of25-50μM. Threeγ-butyrolactone derivatives (57-59) isolated from CMN-1showed quorum-sensingactivity. Among these,59showed the best quorum sensing activity.
Summarily, this work obtained70compounds, including23new compounds, fromfive marine-derived actinomycetes. New compounds (1-2) represent a new condensedpentacyclic skeleton, in which the indole ring is fused with a sesquiterpene moiety.New compound12was a skipped-polyol polyene macrolide, with a tetrahydrofuranketal functionality, which is the first example in skipped-polyol polyene macrolides.New compound53was an aminobenzoic acid glycosides derivative with a rareskeleton. New compounds2and4showed significant antitumor activities, whilecompound7showed strong antimicrobial activities. Quorum sensing activities ofcompound59were reported. This study provides novel structures for marine naturalproduct chemistry and bioactive template for exploring new antitumor andanti-antifungal drugs. Forty-nine compounds, including19new compounds wereobtained from actinomycetes CHQ-64, which was selected by PKSI/NRPS/helogenase gene fragments assays, together with applying activity andchemical screening. All above results show that integrated screening can get moreaimed strains, identify quickly active strain with some biosynthesis gene and findsome compounds with special structures. It is indicated that thegene-activity-chemical integrated screening methods may reduce the high activity butold compounds in the separation of natural products and some extent get more aimedstrains, identify quickly active strain with some biosynthesis gene and find somecompounds with special structures. The results of this study provided someexperimental basis for further in-depth research in the field.
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