《天然药物化学》课程参考文献（海洋天然产物）Biomedicinals from the phytosymbionts of marine invertebrates

Available online at www.sciencedirect.com ScienceDirect METHODS ELSEVIER Methods42(2007)358-376 www.eisevier.com/ocate/ymeth Biomedicinals from the phytosymbionts of marine invertebrates: A molecular approach Walter C.Dunlap,Christopher N.Battershill a,Catherine H.Liptrota, Rosemary E.Cobb,David G.Bourne Marcel Jaspars Paul F.Long,David J.Newman ty of London,Engla nd.UK evelopmental Therapeurics Program.NCI-Frederick.MD.USA Accepted 9 March 2007 Abstract Marine invertebrte animaks suchponge,porgonians,tunicates ad bryoo of biomedicinaly relevant natura umer of wh advancing through Zoan and antho ith host tissues where they reside as extraand intra-cellular symbionts.In some sponges these associated microbes may constitute as much as 40%of the holob ont volume.There is now abundant evidence to suggest t at a significant portion of the bioactive metabolites thought originally to be pro of the source imal are ofn thought to be products derived from ther and marine cyanobacteria are well for and structurally diverse bioactive and condary metabolites suited to drug discovery.Sea sponge cyan teria,and it is sym onts)tha t a duced within the sponge.Accordingly,new collections can be in the field for the presence of phytobionts and,together with PCR primers to identify key polyketide synthase (PKS)and noribosomal peptide synt d b pha tosymbionts of marine organisms. Keywords:Biomedicinals:Marine natural products:Svmbionts:Cva 1.Introduction nic cyanobacteria evolved such for The modern s contain the eatest diversity of life on Earth.and it is where anaerobic life first evolved more eukaryotic algae and plants further advanced the develop than two billion years ago.Within this vast oceanic space, ment of an oxygenie atmosphere the legacy of which now sustains contemporary life.Ancestral metazoans (the hypothetical Urmetazoa)likewise evolved from the oceans utho .Fa+61074772585 2.3 arising from a choanodinoflagellate lineage [4].of which the porifera(sponges)with a fossil record dating

Biomedicinals from the phytosymbionts of marine invertebrates: A molecular approach Walter C. Dunlap a,*, Christopher N. Battershill a , Catherine H. Liptrot a , Rosemary E. Cobb a , David G. Bourne a , Marcel Jaspars b , Paul F. Long c , David J. Newman d a Australian Institute of Marine Science, Townsville, Queensland, Australia b Department of Chemistry, University of Aberdeen, Scotland, UK c School of Pharmacy, University of London, England, UK d Natural Products Branch, Developmental Therapeutics Program, NCI-Frederick, MD, USA Accepted 9 March 2007 Abstract Marine invertebrate animals such as sponges, gorgonians, tunicates and bryozoans are sources of biomedicinally relevant natural products, a small but growing number of which are advancing through clinical trials. Most metazoan and anthozoan species harbour commensal microorganisms that include prokaryotic bacteria, cyanobacteria (blue-green algae), eukaryotic microalgae, and fungi within host tissues where they reside as extra- and intra-cellular symbionts. In some sponges these associated microbes may constitute as much as 40% of the holobiont volume. There is now abundant evidence to suggest that a significant portion of the bioactive metabolites thought originally to be products of the source animal are often synthesized by their symbiotic microbiota. Several anti-cancer metabolites from marine sponges that have progressed to pre-clinical or clinical-trial phases, such as discodermolide, halichondrin B and bryostatin 1, are thought to be products derived from their microbiotic consortia. Freshwater and marine cyanobacteria are well recognised for producing numerous and structurally diverse bioactive and cytotoxic secondary metabolites suited to drug discovery. Sea sponges often contain dominant taxa-specific populations of cyanobacteria, and it is these phytosymbionts (= photosymbionts) that are considered to be the true biogenic source of a number of pharmacologically active polyketides and nonribosomally synthesized peptides produced within the sponge. Accordingly, new collections can be pre-screened in the field for the presence of phytobionts and, together with metagenomic screening using degenerate PCR primers to identify key polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes, afford a biodiscovery rationale based on the therapeutic prospects of phytochemical selection. Additionally, new cloning and biosynthetic expression strategies may provide a sustainable method for the supply of new pharmaceuticals derived from the uncultured phytosymbionts of marine organisms. 2007 Elsevier Inc. All rights reserved. Keywords: Biomedicinals; Marine natural products; Symbionts; Cyanobacteria; Sponges; Polyketides, Nonribosomal peptides; Heterologous expression 1. Introduction The modern oceans contain the greatest diversity of life on Earth, and it is where anaerobic life first evolved more than two billion years ago. Within this vast oceanic space, early forms of photo-oxygenic cyanobacteria evolved such to create the genesis of reduced oxygen necessary for the succession of aerobic metabolism [1]. The evolution of eukaryotic algae and plants further advanced the development of an oxygenic atmosphere, the legacy of which now sustains contemporary life. Ancestral metazoans (the hypothetical Urmetazoa) likewise evolved from the oceans [2,3] arising from a choanodinoflagellate lineage [4], of which the porifera (sponges) with a fossil record dating 1046-2023/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2007.03.001 * Corresponding author. Fax: +61 0 7 4772 5852. E-mail address: w.dunlap@aims.gov.au (W.C. Dunlap). www.elsevier.com/locate/ymeth Methods 42 (2007) 358–376

W.C.Dulap et al.I Methods 4(2007)358-376 359 more than 580 million years ago (the Precambrian)are 2.Bioactive and cytotoxic metabolites from free-living reshwater,estuarine and marine microalgae from oxygen and nutrient exchange to fuel aerobic metab The phytochemistry of microalgal metabolites has olism,since extant forms of symbioses are common to the long been dominated by investigations into the potent toxins produced by harmful phytoplankton blooms nd their endobionts exhibit a high of host gh the ficity and stability that suggests the potential of early trophic food chan,particularly byer-feedingshefish mixtures of biosynthetic congeners.Key representative tances.eates pose serious es pose risks the genera Alexd soning (PSP):okadaic acid (3)and related toxinso Dinophysis and Prorocentrum dinoflagelates are the Man ，of thes shellfish oning (ASP) as (5 and 6) metabolites are potent across a broad spectrum of activi- the dinoflagelate Karenia brevis (formerly Gymnodiniu ties,including antiviral,antibiotic,,and anti rere)cause neurotoxic shellfish poisoning (NSP):cigua cancer d from the notably sponges,tuni Microcystis and Nodularia produce e s been consisten mic ocystins（e.g bioactive metabolites9there is a current surge of inter ates to cause fatal estuarine toxic syn est in the phytochemistry of marine microorganisms for drome. The of harmful microalgal toxins is appreciation largely xtensive and revie 01g toxins was published by Van Dolah [18]and several paper,we exploit the niche idea that marine invertebrates lsrange of m nificance photosynthetic endosymbionts [16].Accordingly, N selecting these phytosymbiotic ass c accumulatior saxitoxin (1) phytoto xins and other we d tion symbioses and host bioaccumulation of phytochemical O H metabolites.We provide some important examples of bio NH Eue Thei Promt Payy H-H search and identify key biosynthetic genes from the cur- 0 ently non-culturable microbial consortia and present e rging technologies or cloning g the biosyntn to achieve a sustainable supply

more than 580 million years ago (the Precambrian) are the most primitive extant animals. Early metazoans may have developed with photosymbiotic partners to benefit from oxygen and nutrient exchange to fuel aerobic metabolism, since extant forms of symbioses are common to the cyanobacteria–sponge assemblage that persist today. It has been noted that the associations between sponges and their endobionts exhibit a high degree of host speci- ficity and stability that suggests the potential of early coevolution between the host sponge and its microbial symbionts [5,6]. Free-living cyanobacteria have been intensely studied in aquatic environments as the progenitors of harmful substances. In eutrophic waters, red tide blooms of cyanobacteria and toxic dinoflagellates pose serious risks to human health from the consumption of seafood contaminated by toxic species. In addition to specific phytotoxins, it is well recognized that genetically diverse marine and freshwater microalgae, including the prokaryotic cyanobacteria, present a valuable resource in the discovery of biomedicinal secondary metabolites. Many of these metabolites are potent across a broad spectrum of activities, including antiviral, antibiotic, antifungal, and anticancer properties of pharmaceutical interest. The ‘‘drugs-from-the-sea’’ effort spanning the last several decades has focused primarily on anti-tumor agents sourced from marine sessile invertebrates, notably sponges, tunicates and bryozoans [7,8]. While there has been consistent effort to screen marine photosynthetic microorganisms for bioactive metabolites [9], there is a current surge of interest in the phytochemistry of marine microorganisms for drug discovery [10–12]. This appreciation owes largely to the realisation that many bioactive metabolites originally attributed to the source animal are actually produced by their microbial consortia [10,13,14]. In this paper, we exploit the niche idea that marine invertebrates are the ‘‘petri dish’’ that sustains a diverse range of microbial life [15], and that phytochemicals of biomedicinal significance can be sourced from animals harbouring photosynthetic endosymbionts [16]. Accordingly, we affirm selecting these phytosymbiotic associations as a selective strategy to optimise performance in the quest to discover novel therapeutics. We present a brief overview of the basic phytochemistry of marine microalgae, including the trophic accumulation of phytotoxins and other bioactive metabolites. We discuss the morphological organisation of microbial-invertebrate symbioses and host bioaccumulation of phytochemical metabolites. We provide some important examples of bioactive metabolites attributed to marine phytosymbionts and argue their potential role in chemical ecology. Additionally, we consider the use of molecular techniques to search and identify key biosynthetic genes from the currently non-culturable microbial consortia and present emerging technologies for cloning the biosynthetic genes for heterologous production of phytochemical metabolites to achieve a sustainable supply. 2. Bioactive and cytotoxic metabolites from free-living freshwater, estuarine and marine microalgae The phytochemistry of microalgal metabolites has long been dominated by investigations into the potent toxins produced by harmful phytoplankton blooms occurring in marine and aquatic environments. Consumption of phytotoxins that accumulate through the trophic food chain, particularly by filter-feeding shellfish, can elicit distinct toxin-specific symptoms of gastrointestinal and neurological illnesses. These phytotoxins are structurally diverse and are often elaborated as complex mixtures of biosynthetic congeners. Key representatives are the saxitoxins (e.g. 1) and gonyautoxins (e.g. 2) from dinoflagellates of the genera Alexandrium, Gymnodinium and Pyrodinium causing paralytic shellfish poisoning (PSP); okadaic acid (3) and related toxins from Dinophysis and Prorocentrum dinoflagelates are the cause of diarrhetic shellfish poisoning (DSP); domoic acid (4) from Pseudo-nitzschia diatoms cause amnesic shellfish poisoning (ASP); brevetoxins (5 and 6) from the dinoflagelate Karenia brevis (formerly Gymnodinium breve) cause neurotoxic shellfish poisoning (NSP); ciguatoxin (7) and maitotoxin (8) from the dinoflagellate Gambierdiscus toxicus are the toxic agents of ciguatera fish poisoning (CFP); cyanobacteria of the genera Anabaena, Microcystis and Nodularia produce potent hepatotoxins, the microcystins (e.g. 9) and nodularins (e.g. 10); and the elusive toxins of Pfiesteria dinoflagellates are reputed to cause fatal estuarine toxic syndrome. The study of harmful microalgal toxins is extensive and has been reviewed elsewhere; an authoritative monograph has been published by UNESCO [17]; a review on the origin and health effects of marine algal toxins was published by Van Dolah [18]; and several overviews on the biochemistry of phytotoxins are available [9,19–21]. HN NH H N H2N H2N O O H NH2 N OH OH saxitoxin (1) NH N N O O N+ NH NH N+ OH OH O H H S O O O H H H S O O O H gonyautoxin-VIII (2) W.C. Dunlap et al. / Methods 42 (2007) 358–376 359

N N N N N HN NH2 Me N O COOH OO OMe O COOH O nodularin-R (10) Notwithstanding toxin production, cyanobacteria are recognized as being one of the most productive groups of microalgae providing a rich and diverse source of bioactive natural products for drug discovery [22–24]. Interesting candidates are as follows. Borophycin (11), related to the boron-containing boromycins, was isolated from Nostoc linkia and N. spongaeforme and has potent cytotoxicity against human epidermoid carcinoma and human colorectal adenocarcinoma cell lines [25,26]. Cryptophycin 1 (12) isolated from Nostoc sp. has shown broad-spectrum cytotoxicity in drug-resistant murine and human solid tumors [27] from which the synthetic analogue cryptophycin 52 (LY355703) (13) was developed by Lilly Research Laboratories for improved therapeutic efficacy, although Phase I trials were ceased due to dose-limiting neurotoxicity [28,29]. A particularly exciting find is cyanovirin (CV-N) (14), a 101 amino acid protein isolated from Nostoc ellipsosporum, that has been placed on an accelerated track for clinical development as a fusion inhibitor of HIV. Cyanovirin has been found to be active against immunodeficiency retroviruses HIV-1, HIV-2, SIV (simian) and FIV (feline) [30], and has high potency against most strains of influenza A and B viruses [31]. Currently CV-N is available as an investigational preparation (vaginal gel) for HIV protection (http:// www.aidsinfo.nih.gov). The potential of these drug candidates to achieve clinical success holds great strategic promise for exploiting the structural complexity of cyanobacterial metabolites across diverse therapeutic areas in future drug discovery [11]. O O O B- O O O O O O OH O O O OH borophycin (11) O O O NH NH O Cl OMe O O O cryptophycin 1 (12) O O NH NH O Cl OMe O O O O cryptophycin 52 (13) 3. Trophic accumulation and chemical ecology of phytochemicals in aquatic and marine environments While the trophic accumulation of microalgal toxins in the aquatic food chain has been intensely studied, less is known of the ecological role and metabolic cost to the algal producer for the biosynthesis of such a vast array of oftencomplex secondary metabolites. It has been noted that organisms of early evolution lacking an immune system are prolific producers of secondary metabolites [32], while others have argued that toxic metabolites are synthesized by microalgal producers primarily to deter predation by zooplankton consumers in a highly competitive environment [33]. Yet, little experimental evidence exists to establish the exact ecological function of most cyanobacterial metabolites. The field of ‘‘chemical ecology’’ attempts to correlate the relational adaptation between the structure and function of secondary metabolites that mediate interactions affecting growth competition, the induction of sexual responses, symbiosis and commensal interactions, predator–prey capture and survival strategies, and a host of other ecology-driven, biochemically mediated, physiological processes [34]. An example of chemical ecology demonstrating a functional environmental adaptation is the well-studied occurrence and distribution of UV-absorbing, mycosporine-like amino acids (MAAs) that occur in micro- and macro-algae, and are trophically accumulated in higher organisms, as a protective sunscreen response to UV exposure [35,36]. In marine ecosystems, recent attention has been given to the role of phytosymbionts within and among invertebrate species, particularly in benthic W.C. Dunlap et al. / Methods 42 (2007) 358–376 361

tropical ecosystems where species diversity and resource competition are high. Accordingly, the role of phytochemicals in tropical symbioses to protect fleshy invertebrate hosts from predation [33] and the accumulation of MAAs (i.e., mycosporine-glycine (15), palythine (16), palythinol (17), and others) in shallow-water invertebrates, including the dinoflagellate-anthozoan symbiosis of reef-building corals [35,36], has been well documented. O H N HO OMe OH COOH mycosporine-glycine (15) H N HO OMe OH HN COOH palythine (16) H N HO OMe OH N COOH HO palythinol (17) One particular source that has proven to be rich for the discovery of novel bioactive metabolites is the diet-derived accumulation of microalgal products in sea hares collected from the Indian Ocean and Japan. The sea hare Dolabella auricularia has yielded an exceptional variety of structurally similar cytotoxic agents putatively derived from their cyanobacterial diet (reviewed extensively by Luesch et al. [37]). The most important of these metabolites are dolastatin 10 (18) and 15 (19) that progressed into clinical trials as anti-cancer mitotic inhibitors, but proved too toxic. However, later generations of these compounds, in particular variations of dolastatin 15, have progressed to clinical examination [38]. Phase II trials of TZT-1027 (soblidotin) (20) have been completed against soft tissue sarcoma (http://www.clinicaltrials.gov/ct/search?term=soblidotin& submit=Search) and phase II trials of ILX 651 (synthadotin or tasidotin) (21) have been completed against melanoma, prostrate and non-small cell lung cancers (http:// www.clinicaltrials.gov/ct/search?term=synthadotin&submit=Search). In addition to several of the dolastatins, dolabellin (22) and the dolastatin-like symplostatin 1 (23) were isolated from cyanobacteria of the genus Symploca, and the dolabellin-like lyngbyabellin A (24) and B (25) were isolated from the cyanophyte Lyngbya majuscula. The sea hare D. auricularia is a generalist herbivore that is presumed to acquire a wide range of cyanobacterial metabolites at low concentrations [39], while selective herbivores such as Stylocheilus longicauda that feed preferentially on the cyanobacterium Lyngbya majuscula bioaccumulate the cytotoxic macrolide aplysiatoxin (26) at high tissue concentrations [40]. Similar cyanobacterial metabolites have been found in filter-feeding sponges. One such example is majusculamide C (27), originally obtained from a deep-water collection of L. majuscula [41] that was re-isolated as a metabolite of the sponge Ptilocaulis trachys [42]. The overarching conclusion of finding such useful cyanobacterial products sequestered by marine invertebrates is that marine cyanobacteria should be regarded as the primary target source of structurally diverse metabolites available for drug discovery, a view long held by others [22,43]. N H N O N O O N O O O H N S N dolastatin 10 (18) N NH N O O N O O N O O N O OMe O dolastatin 15 (19) N H N O N O O N O O O H N TZT-1027 (soblidotin) (20) N NH N O O N O O N O H N ILX 651 (synthadotin or tasidotin) (21) 362 W.C. Dunlap et al. / Methods 42 (2007) 358–376

S N MeO O O O O S N OH OH O Cl Cl dollabellin (22) H N O O N O O N O H N O N N S symplostatin 1 (23) O N O S HN O HN S N HO O O Cl Cl O H O N O S HN O HN S N HO O O Cl Cl O lyngbyabellin A (24) and B (25) O O O O OH O Br OH O OMe OH aplysiatoxin (26) MeO NH NH NH O O O O O O N N NH N NH O O O O O H3C majusculamide C (27) 4. Phytobionts of invertebrate symbioses Marine invertebrates, including sponges, cnidarians and tunicates, often harbor dense and diverse populations of commensal microorganisms that include heterotrophic and chemoautotrophic bacteria, cyanobacteria, fungi, and eukaryotic algae within host tissues where they reside as extra- and intra-cellular symbionts. In some sponges, microbial symbionts may constitute more than 40% of the volume of the holobiont [44] that can be accommodated within specialist bacteriocyte host cells [45]. Singlecell and filamentous cyanobacterial symbionts, belonging to the genera Aphanocapsa, Synochocystis, Phormidium (Oscillatoria), Anabaena and Synechococcus, inhabit the light-exposed pinacoderm (ectosome) of sponges, whereas cyanobacteria found in the mesohyl interior (endosome) are likely concentrated from surrounding seawater by filter feeding. Other important groupings of microalgal-invertebrate symbioses include the obligate Prochloron cyanophytes (Prochlorophyta) of didemnid ascidians (tunicates) and the dinoflagellate-anthozoan symbioses of corals. Dinoflagellate endosymbionts of the genus Symbiodinium (often referred to as zooxanthellae) are common in corals, anemones, jellyfish, some mollusks (especially the giant Tridacna clams) and foraminifera. These remarkable phytobionts drive the formation of coral reefs by capturing sunlight to provide their hosts with photoautotrophic metabolic energy while enhancing skeletal-carbonate deposition by photosynthetic uptake of CO2 [46]. 5. Biomedicinally significant phytochemicals from marine symbioses Marine invertebrates are an abundant source of structurally unique secondary metabolites having proven therapeutic potential, of which a small number are advancing through clinical trials [7,8]. The earliest example was the discovery of arabinose-containing spongouridine (28) and spongothymidine (29) from the Caribbean sponge Cryptotheca (Tethya) crypta [47–49]. These natural products served as a template for the synthesis of the nucleoside antiviral drug Ara-A (Vidarabine) (30) that led to the development of acyclovir (Zovirax) (31), which is active against the herpes virus, and the anti-AIDS drug azidothymidine (AZT) (32) for which Hitchens and Elion were jointly awarded the Nobel Prize in 1988 [8]. Interestingly, Ara-A and spongouridine were later discovered as natural metabolites of the Mediterranean gorgonian Euniicella cavolinin [50]. It is noteworthy that commercial production of Ara-A was obtained by fermentation of Streptomyces griseus [51], providing an early hint that bioactive natural products of marine invertebrates could be of microbial origin (albeit bacterial in this case) accumulated from dietary, commensal or endosymbiotic microorganisms. Among the >18,000 marine natural products (MNPs) described to date, recent reviews give 22 MNPs, or chemically derived analogues, to be in clinical trials [7,52]. The majority of these drug candidates are in trials for anti-cancer treatment (68%) with the remainder in therapeutic areas of inflammation, pain, and asthma, and one candidate for treatment of Alzheimer’s disease. Marine-derived agents under examination for cancer treatment target a broad spectrum of molecular W.C. Dunlap et al. / Methods 42 (2007) 358–376 363

Microbial symbionts have been implicated in the biosynthesis of many bioactive metabolites [54,55] as demonstrated elegantly for theopederin A (37) and onnamide A (38) by isolation of the polyketide synthase genes from the uncultured Pseudomonas sp. bacteriosymbiont of the sponge Theonella swinhoei [56]. However, typically less than 1% of the commensal microbiotic consortia of marine invertebrates are amenable to laboratory culture [57], although significantly higher yields may be achievable (RT Hill, pers. commun.). Even if microbial populations can be successfully separated, undefined metabolic factors from the host are thought to be required for the symbionts to grow ex hospite. Notable exceptions are the microorganisms that produce the telomere-inhibiting griseorhodin A (39) of the rubromycin group of antibiotics. Although first isolated from terrestrial actinomycetes, griseorhodin A was re-discovered from the culture of a symbiotic Streptomyces sp. (strain JP95) from the Australian ascidian Aplidium lenticulum, from which its biosynthetic gene cluster was subsequently cloned, sequenced and biosynthetically expressed in Streptomeyces lividans [58]. Similarly, the anti-infective alkaloid manzamine A (40) was successfully obtained in culture from the bacterium Micromonospora sp. of the deep-water Indonesian sponge Acanthostrongylophora sp. [59,60]. While the biosynthetic pathways for several other marine metabolites have been entirely sequenced [61–63], biosynthetic expression of transgenic metabolites from the uncultured symbionts of marine invertebrates has been elusive despite considerable effort [64,65]. O NH O O O O MeO OH OMe O OH theopederin A (37) O NH O O O NH OH O NH NH2 COOH NH O OMe H MeO OH onnamide A (38) O O O MeO O O OH OH OH O O HO griseorhodin A (39) N N H N H N+ H OH H H manzamine A (40) The New Zealand sponge Mycale hentscheli offers three separate classes of polyketide macrolide metabolites of pharmaceutical interest. Mycalamides A (41) and B (42) were first described as antiviral agents [66] and together with mycalamide D (43) [67] were characterised as eukaryotic cytotoxins that inhibit protein synthesis causing apoptosis [68,69]. Pateamine (44) having immunosuppressive and apoptotic properties [69,70] has been isolated in discrete populations of M. hentscheli that lack mycalamides [71]. Peloruside A (45) was additionally isolated from M. hentscheli as a cytotoxin structurally unrelated to pateamine or the mycalamides [72]. Peloruside A is a 16-membered polyketide macrolide similar to epothilone (46) having potent antimitotic activity with paclitaxel-like microtubule-stabilizing properties that arrest cells in the G2/M phase of the cell cycle [73]. The bioactive metabolite composition of Mycale hentscheli collected from New Zealand is remarkably site-specific, and intra-specific concentrations are variable within regional sites [74,75]. Such chemotypic variation of metabolite synthesis by Mycale argues strongly that sponge conspecifics residing under different abiotic conditions may sustain divergent populations of microbial endobionts, possibly cyanobacterial variants [76], that direct the biosynthesis of these diverse secondary metabolites within a monospecific host. 41 R1 = H; R2 = Me 42 R1 = Me; R2 = Me 43 R1 = H; R2 = H O H N OOO O OR2 OH OR1 OH MeO mycalamides A (41), B (42) and D (43) N S N O O H2N O O pateamine (44) W.C. Dunlap et al. / Methods 42 (2007) 358–376 365

O OMe O O HO MeO OH OHOH O OH peloruside A (45) O OH O OH O S N O epothilone (46) Ascidians (sea squirts) of the family Didemnidae are in obligate symbiosis with Prochloron spp. (unicellular cyanobacteria of the sub-class Prochlorophyta) [77] and contain a variety of bioactive cyclic peptides that belong to at least two groups of metabolites, the octapeptide patellamide family and the heptapeptide lissoclinamide family, plus other non-peptidic metabolites. The patellamide family, including patellamides A–D (47–50), ascidiacyclamide (51), ulithiacyclamide (52) and trunkamide A (53), and the lissoclinamide family, including lissoclinamide 1–5 (54–59) and ulicyclamide (59) are a highly conserved family of oxazoline and thiazole/thiazoline containing cyclopeptides that were obtained from the tropical Indo-Pacific aplousobranch ascidian Lissoclinum patella [78,79]. These compounds have differential cytotoxic properties targeting fibroblast and tumor cell lines [78]. Of the known patellamide and lissoclinamide metabolites, ulithiacyclamide was reported to be the most potent cytotoxic compound with an IC50 (50% inhibition concentration) value of 0.35 lg/ ml for the murine leukemia L1210 cell line [80]. Patellamide D, possibly as a binuclear copper complex [81], has additional pharmacological significance as a selective antagonist for reversing the multidrug resistant CEM/VLB 100 human leukemic cell line towards vinblastine, colchicines and adriamycin treatment [82]. Related hexapeptides were isolated from the ascidian Lissoclinum bistratum also containing Prochloron sp. The hexapeptide bistratamide A (60) and B (61) were found localized in the Prochloron symbionts while the macrocyclic bistramide (bistratene) A (62) and B (63) are attributed to be products of the ascidian [83]. The ascidian Trididemnum solidum contains a symbiotic cyanophyte Synechocystis trididemni closely related to Prochloron sp. [84,85] that is proposed to produce several cyclic depsipeptides, of which didemnin B (64) having an LC50 (50% lethal concentration) value of 0.001 lg/ml against L12310 leukemia cells was the first to enter clinical trials as a marine-derived antineoplastic agent [86], although suspended later in Phase II clinical trial for acute toxicity [87]. NH S O N N NH O N NH S N NH R1 O R3 O O R2 R4 O (47) Patellamide A R1=R3=CHMe2 R2=R4=CHMeEt Ring 1-no Me (48) Patellamide B R1=CH2Ph R2=CH2CHMe2 R3=Me R4=CHMeEt (49) Patellamide C R1=CH2Ph R2=CHMe2 R3=Me R4=CHMeEt (50) Patellamide D R1=CH2Ph R2=R4=CHMeEt R3=Me (51) Ascidiacyclamide R1=R3=CHMe2 R2=R4=CHMeEt 1 patellamides A-D (47-50) and ascidiacyclamide (51) NH O N N S R2 O HN O N O NH N S O R1 O NH S S (52) Ulithiacyclamide A R1=R2=CH2CHMe2 ulithiacyclamide (52) N N H O N S HN O O NH O O NH O O O trunkamide A (53) 366 W.C. Dunlap et al. / Methods 42 (2007) 358–376

NH S O N N N NH O S N NH O R2 O R1 O X (54) Lissoclinamide 1 R1=CHMe2 R2=CHMeEt a=L b=D X=thiazole (Δ) (55) Lissoclinamide 2 R1=CHMeEt R2=CHMe2 a=D b=D X=thiazoline (56) Lissoclinamide 3 R1=CHMeEt R2=CHMe2 a=D b=L X=thiazoline (57) Lissoclinamide 4 R1=CHMe2 R2=CH2Ph a=L b=D X=thiazoline (58) Lissoclinamide 5 R1=CHMe2 R2=CH2Ph a=L b=D X=thiazole (Δ) (59) Ulicyclamide R1=CHMeEt R2=Me a=L b=D X=thiazole (Δ) a b Δ lissoclinamide 1-5 (54-58) and ulicyclamide (59) N N S O N N N O S N O O Δ A = thiazoline B = thiazole (Δ) bistratamide A (60) and B (61) O N N O O HO OH O O O bistratene (bistramide) A (62) O N N O O O O OH O OH bistramide B (63) Soft corals are known to be the ‘‘producers’’ of pharmacologically important terpenes, the most developed of which are the potent anti-inflammatory pseudopterosins A–D (65–68) isolated from the gorgonian (sea whip) coral Pseudopterogorgia elisabethae and are used in the Estee Lauder’s Resilience skin care products [88]. The biosynthetic source of these diterpene glycosides has been identi- fied to be the dinoflagellate Symbiodinium sp. partner [89], although reputedly derived from bacteria associated with the Symbiodinium endosymbiont [10]. Recently the full biosynthetic pathway has been elucidated for the pseudopterosins with elisabethatriene (69) as a key intermediate [90,91]. Two other marine-derived non-steroidal anti-inflammatory terpenoids are the 5-lipoxygenase inhibitor fuscoside (70) from the Caribbean soft coral Eunicea fusca [92,93] and the phospholipase A2 inhibitor manoalide (71) from the marine sponge Luffariella variabilis [94,95]. As with the pseudopterosins, it has been demonstrated that fuscol (72) from E. fusca is biosynthesised by its dinoflagellate partner [96], whereas the biosynthetic origin of manoalide in L. variabilis, which lacks resident phytosymbionts, is uncertain. The diterpene glycoside eleutherobin (73) isolated from the soft coral Eleutherobia sp. from Western Australia is a potent antimitotic cancer cell inhibitor with an IC50 (10–15 nM) similar to that of paclitaxel [97]. Eleutherobin, together with several related derivatives, was later discovered in the encrusting coral Erythropodium caribaeorum inhabiting waters of South Florida and the Caribbean [98]. The finding of eleutherobin in such diverse taxa, although unproven, argues strongly that this pharmacophore is produced by a microbial symbiont common to both corals. It should be noted that eleutherobin, however, is an isolation artefact [99] that occurs from methanol extraction of the natural C-4 hemiacetal precursor, desmethyleleutherobin (74). 6. Targeting phytosymbiont selection in drug bioprospecting using molecular discrimination As discussed previously, MNPs that co-occur in unrelated genera of marine organisms, or are closely related to secondary metabolites of unambiguous microbial origin, are likely to be derived from dietary sources or from commensal microbiota residing within an invertebrate host. The remarkable site-specific divergence in metabolite biosynthesis of the mycalamides (41–43), pateamine (44), and peloruside (45) by New Zealand Mycale conspecifics [75], although yet to be substantiated, argues strongly that the sponge residing under different abiotic conditions sustains different chemotypic populations of endosymbionts, possibly involving commensal cyanobacteria [76]. NH OH O O O O HN O N O N Me OMe O O O N H O Me N O N O OH didemnin B (64) W.C. Dunlap et al. / Methods 42 (2007) 358–376 367

《天然药物化学》课程参考文献（海洋天然产物）Biomedicinals from the phytosymbionts of marine invertebrates - A molecular approach