O. V. Mosin

M.V. Lomonosov Moscow State Academy of fine Chemical Technology, Moscow 153233.

Blue-green algae cyanobactcria are among the most primitive of organisms, having an evolutionary history of about four billion years. These procaryotic, photosynthetic microorganisms, which have the morphology and physiology of both bacteria and plants, are ubiquitous and have been adapted to a wide variety of environments, including hot springs and polar ice caps. Although the most conspicuous species are found in freshwater and terrestrial habitats, it is estimated that the total number of autonomous marine species is more than double that of strictly terrestrial ones.

The first blue-green algae to be chemically investigated were the toxic species that frequently bloom in eutrophic lakes and reservoirs and account for animal and human intoxications. Neurotoxicity and hepatotoxicity arc the most common types of poisoning. Strains of Anabaena flosaquae, a filamentous species that elaborates predominantly neurotoxic alkaloids (anatoxins) (Devlin et al. 1977; Carmichael, Gorham, 1978), and strains of Microcystis aeruginosa, a colonial species that produces hepatotoxic cyclic heptapeptides (Bishop et al., 1959; Rinehart et al., 1988), are responsible for most of the toxic illnesses. As more and more water supplies become eutrophic, there is growing concern about the effects of toxic algal blooms on the quality and safety of water that is used for human, as well as wildlife and domestic animal, consumption. In a recent case of human poisoning in Australia (Falconer et al., 1983), for example, hepatotoxicity coincided with the appearance of a toxic bloom of M. aeruginosa in the drinking-water supply. In developing countries such as Chine and, cyanobactcrial poisoning appears to be playing a significant role in the markedly higher incidence of human liver cancer in areas that are heavily dependent on surface drinking-water (Yu, 1989; Nishiwaki-Malsushimaetal, 1992).

According to a review (Baslow, 1977) that blue-green algae contain antibiotics, growth stimulants, and carcinogens in addition to potent toxins, the scientists began an intensive study of thismicroorganisms as a source of new pharmaceuticals and agrochemicals (Moore ct al., 1988).

Several toxins from blue-green algae have been found to be important tools for pharmacological research. The aplysiatoxins (Kato, Seheuer, 1975; Mynderse et al., 1977) and lyngbyatoxins (Cardellina et al., 1979), highly inflammatory acetogenins and alkaloids, respectively, associated with a marine blue-green alga Lyngbya majuscula that is responsible for a severe contact dermatitis in Hawaii (Sedula et al., 1982), are potent activators of protein kinasc С and powerful tumor promoters, having mechanisms of action that are essentially identical with those of the phorbol esters (Fujiki et al., 1990; Fujiki, Suganuma, 1995).

The microcystins (Botes et al., 1984, 1985; Carmichael et al., 1988) and potent inhibitors of protein-serine/threonine phosphatases 1 and 2A (MacKintosh et al,, 1990; Honkanen et al., 1990), exhibiting exactly the same effects at the molecular level as okadaic acid. At the cellular and whole animal levels, however, the microcyslins and okadaic acid respond very differently. Okadaic acid is able to penetrate cellular membranes to inhibit protein phosphatases located inside the cells; however, microcystins are not able to get inside cells. Okadaic acid is a potent inflammatory agent that is responsible for diarrhetic shellfish poisoning; however, it is not hepatotoxic.

Microcystins are among the most hepatotoxins known, but under normal circumstances, they do not appear to possess inflammatory or diarrhea-producing activities. Nodularin, a microcystin-related cyclic pentapeptide associated with Nodularia spumigena, the first blue-green alga to be implicated in animal poisoning (Francis, 1878), exhibits similar biological effects (Honkanen et al., 1991). Microcystins are tumor-promoting while nodularin is carcinogenic (Ohta et al., 1994). Clearly cyanobactcrial toxins have become very important new probes for the study of cellular regulation and other biological processes.

A large number of extracts of blue-green algae show broad-spectrum fungicidal activity, but not broad-spectrum antibacterial activity. Extracts of more than 1500 strains representing some 400 species of blue-green algae have been screened and nine percent of the extracts have been found to be antifungal at 1 mg/disc against one or more test organisms, viz. Aspergillus oryzae, Candida albicans, Penicillium notatum, Saccharomyces cerevisiae, and Trickophyton mentagrophytes (R. E. Moore, G. M. L. Patterson, unpublished). For most of the extracts of cyanophytes belonging to the Stigonemataceae, indole alkaloids that possess isonitrile groups such as the hapalindoles (Mooreetal., 1987), fischerindoles (Parket al., 1992), ambiguincs (Smitka et al., 1992), and welwitindolinones (Stratmann et al., 1994) are responsible for the antifungal activity.

A fungicide with potential utility in the agricultural area is majusculamide C, a cyclic nonadepsipeptide from a deep-water variety of the marine cyanophytc Lyngbya majuscula (Carter et al., 1984).

Potent activity was observed against a broad-spectrum of fungal plant pathogens, including resistant strains, such as Phytophthora infestans, the causative organism of tomato late blight, and Plasmoporaviticola, the causative organism of grape downy mildew (Moore, Mynderse, 1982).

Blue-green algae have been found to be excellent sources of new anticancer agents (Patterson et al., 1991a; Gerwick et al., 1994). Research has shown that a relatively high percentage of extracts of blue-greens collected in the field arc active in vivo, but often the cyanophytes, specifically those of marine origin, can not be recollected in sufficient quantities or with the same activities for isolation, identification, and biological evaluation of the agents responsible for the antineoplastic activity (Moore, 1982). Six percent of the extracts of over 2000 cultured strains, however, were found to be cytotoxic against human tumor cell lines at 20 mg/mL (Patterson et al., 1991b). Less than 1 % of the extracts, however, were solid tumor selective (Valerioteetal., 1994) and/or tumor selective, but several of these showed equal cytotoxicily against drug sensitive and drug resistant cell lines. Furthermore, some of the non-cytotoxic extracts (<1%) showed significant multiple-drug-rcsistance (MDR) reversing activity.

In contrast to field-collected blue-greens, almost all of the cultured blue-greens having anticancer activity proved to be terrestrial and freshwater species.

The most literature on secondary metabolites from blue-green algae published prior to 1980 has been comprehensively reviewed by Moore, 1981.

Peptides and Depsipeptides

Most of the secondary metabolites isolated from blue-green algae are peptides and depsipeptides and the majority of these are cyclic (Moore, 1995).

The most familiar peplides are the microcystins which belong to a class of cyclic heptapeptides comprised of approximately 50 members (Rinehart et al., 1994). Several of the microcystins have the general structure cyc-[D-Ala-L-X-D-erythro-methy\-isoAsp-L-F-Adda-D-isoGlu-rnethyldehydro-Ala where X and Y are variable L-amino acids is a unique amino acid (2S,3S,8S,95)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-(4,6)-decadienoic acid (Namikoshi et al., 1989). For example, X and Y in microcystin-LR, the major hcpatotoxin in most strains of M. aeruginosa from the Northern Hemisphere, are leucine and arginine. The structurally-related cyclic pentapcptide, nodularin, lacks the D-Ala and L-Leu units found in microcystin-LR. Acyclic peptides related to microcystins and nodularin have also been isolated (Choi et al., 1993). The biosynthesis of microcystin-LR has been studied (Moore et al., 1991).

Calophycin, a potent broad-spectrum fungicide from Calothrixfusca EU-10-1 (Moon et al,, 1992), is a cyclic decapeptide containing a novel palmitic acid unit. The unusual unit has also been identified in puwainaphycin E, one of a family of cyclic decapcptides from Anabaena sp. BQ-16-1 (Gregson et al., 1992). The laxaphycins are a large family of cyclic undeca- and dodecapeptides, the major representative of each class being laxaphycins A and B, respectively, which together act synergistically to produce the antifungal activity of the crude extract of Anabaena (Frankmolle etal., 1992).

The laxaphycins closely resemble a group of cyclic peptides known as the hormothamnins that have been isolated from the marine cyanophyte Hormothamnion enteromorphoides (Gerwick et al., 1992). Scytonemin A is a cyclic undccapeptide from Scytonema sp. U-3-3 that possesses an unusual p-amino acid unit and three substituted prolines (Helms et al., 1988).

The tantazoles are modified hexapeptides from the terrestrial cyanophyte Scytonemamirabile BY-8-1. All of the compounds possess a sequence of four contiguous cysteine-derived A imiazoline rings attached 4,2 to one another with an isopropyl group connected to C-2 of the first thiazoline ring (ring A) and a threonine-derived oxazole ring (ring E) attached to C-4 of the fourth thiazoline (ring D) via C-2. A glycine-derived appendage is linked to C-4 of the oxazole ring. Tantazole В is a solid tumor selective cyloloxin (Carmeli et al., 1993). Structurally-related mirabazoles lack the oxazole ring (CaimelietaL, 1991).

Westicllamide is a modified cyclic hexapeptide from Westiellopsis prolifica (Prinsep et al., 1992). Its isolation from a blue-green alga provides circumstantial evidence for algal symbionts (Prochloron spp.) playing a role in the biosynthesis of closely-related cyclic peptidcs found in marine tunicates, e.g. the bistratamides in Lissodinum bistratum (Dcgnan et al., 1989). Similar pcptides (e.g. dolastatin 3) found in marine molluscs undoubtedly have a cyanobactcrial origin (Pettit ct al., 1987).

The cryptophycins form the largest depsipeplide class (Golakoti et al., 1995). Structurally most cryptophycins consist of a 6-hydroxy acid unit (A), an a-amino acid unit (B), amino acid unit (C), and an a-hydroxy acid unit (D), connected together in an ABCD sequence. Total syntheses of cryptophycin-1, -3 and -4 have been achieved (Barrow et al., 1995). Cryptophycin-24 from Nosloc sp. GSV 224 is identical with a marine sponge constituent, arenastatin A (Kobayashi et al., 1994).

Several biologically-active cyclic depsipcptides have been isolated from Microcystis spp., viz. a tyrosinase inhibitor, microviridin (Ishitsuka et al., 1990); the plasmin and trypsin inhibitors, micropeptins A and В (Okino et al., 1993a) and 90 (Ishida etal., 1995);acell-differentiation-promoler,microcystilidcA(Tsukamotoctal., 1993); the acruginopeplins (Haradaetal., 1993); and the cyanopeptolins (Martinet al., 1993). These depsipeptidcs and a serine protcinase inhibitor, A90720A, from Microchaete loktakensis IC-39-2 (Lee et al., 1994) possess an unusual 3-amino-6-hydroxy-2-piperidone unit that was first described in dolastatin 13, one of the cytotoxins found in the mollusc Dollabella auricularia (Pettit ct al., 1989). The total structure of A90720A was elucidated by X-ray crystallography of the bovine trypsin-A90720A complex. Hapalosin is a multidrug-resistance reversing agent from Hapalosiphon welwitschii IC-52-3 (Slratmann ct al., 1994a).

The angiotensin-converting enzyme inhibitor microginin (Okino et al., 1993) and the thrombin and trypsin inhibitors aeruginosms 298-A (Murakami et al., 1994) and 98-A and В (Murakami et al., 1995) are acyclic peptides. Majusculamide D (Moore, Entzeroth, 1988) and microcolins A and В are immunosuppresive, acyclic N-acyl pyrrol in-ones where the acyl group is a peptidal group. Mirabimides A-D (Carmeli et al., 1991a) are cytotoxic, acyclic N-acyl pyrrolinones where the acyl group is a depsipcptidal group.

Alkaloids

Anatoxin (Matsunaga et al., 1989), a unique arginine-derived (Hemscheidt et al., 1995) phosphate ester of a cyclic N-hydroxyguanidine and the most potent anticholinesterase known, is one of the neurotoxic alkaloids produced by Anabaena flosaquae. Cylindrospermopsin (Ohtani et al., 1992), a cyclic guanidine, is a potent hepatotoxic alkaloid associated with a strain of Cylindrospermopsis raciborskii that was implicated in an outbreak of hepatocnteritis on Palm Island in northern Queensland, Australia in 1979. Homoanatoxm-a (Skulberg et al., 1992) is a bicyclic neurotoxic alkaloid associated with Oscillaloriaagardhii NOF-81. Besides the paralytic shellfish poisons, neosaxitoxin and saxitoxin, Aphanizomenon flosaquae elaborates a novel tricyclic alkaloid, aphanorphine (8-hydroxy-l,3-dimcthyl-2,3,4,5-tetrahydro-l,4-methano-3-bcnz-azepinc) (Gulavitaetal., 1989).

Lyngbyatoxin A is the major indole alkaloid associated with dermatitis-producing Lyngbya majuscula and is identical with the fermentation product teleocidin A-l (Sakai et al., 1986). The arginine vasopressin inhibitor hapalindolinone A (Schwartz, 1987) and the MDR-reversing agent N-methylwelwitindolinone С isocyanate (Stratmann et al., 1994) are examples of the large number of indole alkaloids found in the family Stigonemataceae. Scylonemin (Proteau et al., 1993) is a dimeric indole alkaloid that functions as an effective ultraviolet sunscreen pigment in the sheaths of cyanobactcria. Cytotoxic, antiviral indolocarbazoles have been isolated from Nostoc sphaericum EX-5-1 (Kniibel et al., 1990) and the tjipanazoles are antifungal indolo[2,3-a]carbazolc alkaloids from Tolypothrix tjipanasensis DB-1-1 (Bonjouklian et al., 1991). The bauerines are antiviral carbolines from Dichothrixbaueriana GO-25-2 (Larsen et al., 1994).

Aulosirazole (Stratmann et al., 1991b), a naphtho[2,3-disothiazole alkaloid from Aulosira DO-8-1), is a solid-tumor selective cytotoxin. Curacin A (Nagle et al., 1995), a cyclopropane-containing thiazole alkaloid from Lyngbya majuscula, is an inhibitor of microtubule assembly and the binding of colchicine to tubulin. Mirabimide E (Paiket al., 1994), a N-acylpyrrolinone from Scytonemamirabile BY-8-1, is a solid tumor selective cytotoxin. Tolyporphin is a novel multi-drug resistance reversing porphyrin from the blue-green alga Tolypothrix nodosa HT-58-2 (Prinsep et al., 1992a). Unusual nucleosides such as tubercidin and toyocamycin (Stewart et al., 1988) and 9-deazaadenosine (Namikoshi et al., 1993) sometimes account for potent cytotoxicity associated with extracts of Nostocaceae.

Polyketides

Acutiphycins (Barchi et al., 1984) are cytotoxins from Oscillatoriaacutissima B-l. Mirabilene isonitriles (Carmeli et al., 1990) are cytotoxic, acetogenic constituents of Scytonemamirabile ВY-8-1. Oscillatoxins (Moore et al., 1984; Entzeroth et al., 1985) are inflammatory agents that are structurally and biologically related to the aplysiatoxins. Acetogenic nostocyclophanes from Nostoc linckia В1932 and cylindrocyclophanes from Cylindrospermum licheni/orme ATCC 29204 are presently the only natural paracyclophanes (Moore et al., 1990; Bobzin, Moore, 1993). Borophycin is a complex of boric acid from Nostoc linckia GA-5-23 (Hemscheidt et al., 1994) that is related to the fermentation products boromycin and aplasmomycin. New malyngamides have been isolated from Lyngbya majuscula (Ainslie et al., 1985).

The scytophycins (Ishibashi et al., 1986) and tolytoxin (Carmeli et al., 1993a) are cytotoxic polyketides that have been isolated from several blue-greens belonging to the Scytonemataceae. Structurally-related swinholides found in the sponge Theonella swinhoei are believed to be produced by a cyanobacterial symbiont(Kitagawactal., 1990).

Carbohydrates

An unusual O-acetyl-O-butyryl-O-carbamoyl-O,O-dimethyl-a-cyclodextrin has been found in Tolypothrix byssoidea (Moore et al., 1986).

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