Controlling production of brominated cyclic depsipeptides by Pseudoalteromonas maricaloris KMM 636T.

AIMS: This study aims at evaluating the impact of the nutrient medium components on the in vitro production of the cytotoxic alterochromides. METHODS AND RESULTS: The employment matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) facilitated the identification of a range of brominated cyclic depsipeptides with molecular masses of 843/845, 857/859 and 922/924/926 Da, and 936/938/940 Da produced by the marine bacterium Pseudoalteromonas maricaloris KMM 636T. The fractions of cytotoxic yellow pigments yielded after methanol extraction of P. maricaloris KMM 636T cells grown on five nutrient media were solely composed of brominated cyclic depsipeptides. Bromo-alterochromides A and B were obtained after cultivation on low nutrient media, while dibrominated derivatives were the principal components of the biosynthesis during cultivation on nutrient rich media. CONCLUSIONS: The quantity of bromo-alterochromides and their dibromo- derivates varied depending on the media composition. SIGNIFICANCE AND IMPACT OF THE STUDY: MALDI-TOF mass spectrometry enables to generate accurate mass analysis for the identification of peptide and its derivates which is important in controlling the production of biologically active compounds in vitro.

New diterpenic altrosides of the fungus Acremonium striatisporum isolated from a sea cucumber.

Two new diterpenic glycosides, virescenosides M (1) and N (2), have been isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Their structures were determined on the basis of MS and NMR data as beta-D-altropyranosido-19-7-oxo-isopimara-8,15-diene-2alp ha, 3beta-diol (1) and beta-D-altropyranosido-19-isopimara-7, 15-diene-2alpha,3beta,6beta-triol (2). Three other altrosides (3-5), identified as virescenosides A, B, and C from the terrestrial strain Acremonium luzulae, were also isolated. The cytotoxic activity of the virescenosides was examined.

Cytotoxicity of natural ginseng glycosides and semisynthetic analogues.

The cytotoxicity of natural glycosides from Ginseng, semisynthetic analogues and related triterpenes of the dammarane series, isolated from the leaves of the Far-East species of the genus Betula was studied in order to elucidate structure-activity relationships. Some of the compounds studied were active against the human lung carcinoma GLC4 and adenocarcinoma COLO 320 cell lines. The natural glycosides displayed the lowest cytotoxicity. The triterpenes of the dammarane series used as starting aglycones for semisynthetic derivatives were moderately cytotoxic. The dammarane triterpenes possessing keto groups and their semisynthetic glucosides were the most active compounds tested. Cytotoxic effects of the dammarane glucosides were inversely proportional both to the number of sugars attached to the aglycones and to the number of hydroxy groups of the aglycones. The type of side chain and the configuration of the hydroxy group at C-3 in aglycones did not have a significant influence on the cytotoxicity.

Synthesis of some hydroxynaphthazarins and their cardioprotective effects under ischemia-reperfusion in vivo.

A series of hydroxynaphthazarins has been synthesized. Some of them were found in in vivo experiments to be protectors of myocardium under ischemia-reperfusion and to reduce the infarction zone by 50% without any adverse effect. All compounds exhibit a moderate or small toxicity and are active in low doses.

Synthesis of ginsenoside Rg3, a minor constituent of Ginseng radix.

Glycosylation of 12beta-acetoxy-dammar-24-en-3beta,20(S)-diol (4), with hepta-O-acetyl-alpha-sophorosyl bromide (5) under catalysis by Ag2CO3 or Ag2O afforded a chromatographically unseparated mixture of the alpha- and beta-linked octaacetates 6 and 7 in an approximately 2.5:1 ratio. After deprotection and chromatographic purification, the free alpha- (8) and beta-glycosides (9) were obtained. Sophoroside 9 was identical in all respects with ginsenoside Rg3, the minor component of Ginseng Radix rubra. All compounds were fully characterized by 1H and 13C NMR spectroscopy.

Modification of carboxyl groups in sea anemone toxin RTX-III from Radianthus macrodactylus.

The toxin was treated with [14C]trimethyloxonium tetrafluoroborate or [3H]glycine methyl ester in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide. Esterification of separate carboxyl groups with [14C]trimethyloxonium tetrafluoroborate decreased the toxicity no more than two-fold. Blocking of any single carboxyl group with [3H]glycine methyl ester did not cause more than a two-fold decrease of toxicity, and modification of two carboxyl groups caused no more than a six-fold decrease. Partial localization of modified residues in the amino acid sequence was performed. By circular dichroism, it was shown that the decrease of toxicity was not associated with alteration of secondary or tertiary structure. It is concluded that free carboxyl groups are not absolutely essential for toxicity, however they are necessary for expression of the maximum RTX-III toxicity.

Modification of arginine in sea anemone toxin RTX-III from Radianthus macrodactylus.

Chemical modifications of the polypeptide neurotoxin RTX-III have allowed us to study the functional role of Arg residues. The effect of chemical modification has been estimated by measuring toxicity in mice. 2,4-Pentanedione did not react with Arg residues of RTX-III even after 100 hr incubation. Malonic aldehyde reacted readily with RTX-III, yielding an unusual derivative; a Schiff's base obtained by condensation of one of two aldehyde groups of malonic aldehyde with the guanidine group. The derivative was one-fourth as toxic as the native toxin. Modification of the guanidine side chain of Arg-13 with both 1,2-cyclohexanedione and phenylglyoxal decreased the toxicity of RTX-III by a factor of five. We conclude that Arg-13 is not fully responsible for toxicity. The toxin-receptor attachment might be multipoint, involving several structural elements of the protein molecule, with Arg-13, probably being one of them. The guanidine side chain of Arg-45 is buried in the sequence and apparently functionally nonessential.

Triterpene glycosides from sea cucumber Stichopus japonicus Selenka.

Triterpenoidal oligoglycosides from the far eastern sea cucumber Stitchopus japonicus Selenka collected in the Posiet Bay, Japan Sea were investigated. The glycoside fraction was shown to consist of two main components, holotoxins A1 and B1 which have been isolated by chromatography on silica gel columns. The chemical structures of the holotoxins were elucidated on the basis of chemical and physiochemical evidence (for formula see text). The proposed structures were compared with close related but not identical structures of holotoxin A and B from other collection of the same holothurian. The probable reasons of the distinction were discussed. The previously-established difference in glycoside structures between Stichopus japonicus and other representatives of the family Stichopodidae was corroborated.

Steroids in Porifera. II. Steroid derivatives from two sponges of the family Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern of side chain alkylation.

A trisulfated derivative of 24,25,26,26-tetramethyl-5 alpha-cholest-23E-ene-2 alpha, 3 beta, 6 alpha-triol (sokotrasterol sulfate) has been isolated from the sponge Halichondriidae gen. sp., collected near Sokotra Island (Arabian Sea), and its structure has been elucidated. The side chain of the new steroid involves a "normal" alkylation at C-24 and the unprecedented addition of two extra methyl groups at C-26 and one extra methyl group at C-25. A free sterol fraction contained only 24-isopropyl-5-cholesten-3 beta-ol and 24-isopropyl-5, 22E-cholestadien-3 beta-ol. 24-Isopropyl-5, 22E-cholestadien-3 beta-ol as sole monohydroxy sterol and halistanol sulfate as major polyhydroxylated steroid derivative have been detected in Halichondria sp., a Madagascar sponge.

A comparative study in vitro of physiological activity of triterpene glycosides of marine invertebrates of echinoderm type.

1. The cytostatic and antimicrobial activity of triterpene glycosides of 19 holothurian species of the Pacific tropical zone has been studied. 2. It has been demonstrated that yeast and tumor cells display a comparable sensibility to the action of triterpene oligosides of sea cucumbers. 3. Gram-positive and Gram-negative bacteria are not sensible to the action of glycosides in doses to 500 mkg/ml. 4. Triterpene glycosides-stichoposides, thelothurins and oligosides of Holothuria of genus Bohadschia are the most active in relation to fungal, yeast microflora and tumor cells.

Effect of marine glycosides on adenosinetriphosphatase activity.

Marine glycosides from the sea cucumbers Actinopyga agassizi, Holothuria atra, Bohadschia argus, Cucumaria fraudatrix, Astichopus multifidus and Thelenota ananas inhibit both Na+-K+ ATPase and Mg2+-ATPase of rat brain in vitro. The glycoside-cholesterol complex of these compounds does not influence ATPase activity. Asterosaponins from starfishes Linckia guildingi and Linckia laevigata possess a slight inhibiting effect. The triterpene glycosides from sea cucumbers are more powerful inhibitors than steroidal glycosides from starfishes.