John K. MacLeod

Purification and characterization of ciguatoxins from moray eel (Lycodontis javanicus, Muraenidae)

Viscera (48.3 kg) from moray eels (Lycodontis javanicus) collected in a ciguatera endemic area were extracted and the ciguatoxins characterized. Three major ciguatoxins, CTX-1, CTX-2 and CTX-3, were isolated and purified to homogeneity on reverse phase high performance liquid chromatography. Several minor toxins were also detected. CTX-1 (490 micrograms) was comparable by both 1H nuclear magnetic resonance (1H NMR) and mass spectroscopy (MH+ m/z = 1111) to ciguatoxin isolated previously from moray eels. CTX-2 (280 micrograms) and CTX-3 (100 micrograms) were less polar ciguatoxins not previously characterized. CTX-2 and CTX-3 differed from CTX-1 by 16 mass units, suggesting that they were less oxygenated analogues. 1H NMR revealed that the hydroxyl at C54 in CTX-1 was absent in CTX-2 and CTX-3. An additional change in the chemistry of CTX-2 compared to CTX-1 and CTX-3 was also suggested on the basis of 1H NMR, indicating that CTX-2 may arise from a different precursor to CTX-1. CTX-3 is likely to be an intermediate in the oxidation of a gambiertoxin (sodium channel toxins from Gambierdiscus toxicus) to CTX-1. The i.p. LD50 values for CTX-1, CTX-2 and CTX-3 were 0.25, 2.3 and 0.9 micrograms/kg, respectively. The signs induced in mice by the ciguatoxins were similar, except that CTX-2 and CTX-3 induced hind-limb paralysis that was absent with CTX-1. Each ciguatoxin was potent orally. CTX-1, CTX-2 and CTX-3 competitively inhibited the binding of [3H]brevetoxin-3 to voltage-dependent sodium channels with relative potencies qualitatively (but not quantitatively) comparable to mouse lethality. This study reveals that the relatively small chemical differences between CTX-1, CTX-2 and CTX-3 give rise to significant structure-activity and pharmacokinetic differences.

Detection of cytokinins in a seaweed extract

Abstract trans-Zeatin, trans-zeatin riboside, their dihydro derivatives, isopentenyladenine and isopentenyladenosine have been identified and quantified in Seasol, a commercial extract of Tasmanian Giant Bull kelp, Durvillea potatorum.

Toxic tetranortriterpenes of the fruit of Melia azedarach

Abstract Four new tetranortriterpenes, meliatoxins A1, A2, B1 and B2 have been isolated and identified as toxic constituents of the fruit of Melia azedarach L. var. australasica. Toxicity and pathological results confirm that the meliatoxins are responsible for most but not all of the symptoms resulting from the ingestion of whole fruit.

Regulators of cell division in plant tissues : XXV. Metabolism of zeatin by lupin seedlings.

Abstract[3H]zeatin was supplied through the transpiration stream to de-rooted lupin (Lupinus angustifolius L.) seedlings. The following previously known metabolites were identified chromatographically: 5′-phosphates of zeatin riboside and dihydrozeatin riboside, adenosine-5′-phosphate, zeatin riboside, zeatin-7-glucopyranoside, zeatin-9-glucopyranoside, adenine, adenosine and dihydrozeatin. Five new metabolites were purified; four of these contain an intact zeatin moiety. Two were identified unequivocally, one as l-β-[6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purin-9-yl]alanine, a metabolite now termed lupinic acid, and the second as O-β-d-glucopyranosylzeatin. These two compounds were the major metabolites formed when zeatin solution (100 μM) was supplied to the de-rooted seedlings. The radioactivity in the xylem sap of intact seedlings, supplied with [3H]zeatin via the roots, was largely due to zeatin, dihydrozeatin and zeatin riboside. When [3H]zeatin (5 μM) was supplied via the transpiration stream to de-rooted Lupinus luteus L. seedlings, the principal metabolite in the lamina was adenosine, while in the stem nucleotides of zeatin and adenine were the dominant metabolites. O-Glucosylzeatin and lupinic acid were also detected as metabolites. The level of the latter varied greatly in the tissues of the shoot, and was greatest in the lower region of the stem and in the expanding lamina. Minor metabolites also detected chromatographically were: (a) dihydrolupinic acid, (b) a partially characterized metabolite which appears to be a 9-substituted adenine (also formed in L. angustifolius), (c) glucosides of zeatin riboside and/or dihydrozeatin riboside, and (d) O-glucosyldihydrozeatin. While lupinic acid supplied exogenously to L. luteus leaves underwent little metabolism, chromatographic studies indicated that O-glucosylzeatin was converted to its riboside, the principal metabolite formed, and also to adenosine, zeatin and dihydrozeatin. A thinlayer chromatography procedure for separating zeatin, dihydrozeatin, zeatin riboside and dihydrozeatin riboside is described.

Regulators of cell division in plant tissues: XXIII. The identity of an unusual metabolite of 6-benzylaminopurine☆

When the cytokinin 6-benzylaminopurine was supplied to de-rooted radish seedings, the principal metabolites formed were the 7- and 9-glucosides. However the cytokinin activity of these glucosides was much less than that of a minor metabolite. This metabolite was purified (yield 550 mug from 40 600 seedings), identified as 6-benzylamino-3beta-D-glucopyranosylpurine and synthesized. It is the first compound with a glycosidic linkage at position 3 of a purine ring to be isolated from a plant tissue.

Identification of cytokinin glucosides in a seaweed extract

Zeatin-O-glucoside, its dihydro derivative, and dihydrozeatin riboside-O-glucoside have been identified as the main cytokinin-O-glucosides in Seasol, a commercial preparation from Tasmanian giant bull kelp marketed as a liquid organic fertilizer. The analysis, which also indicated the presence of zeatin riboside-O-glucoside, was carried out by gas chromatography-mass spectrometry of the aglucones using the stable isotope dilution method.

The trikentrins : novel indoles from the sponge trikentrion flabelliforme

Abstract Five new indoles, cis -trikentrin A (1), trans -trikentrin A (2), trans -trikentrin B (3), cis -trikentrin B (4) and iso - trans -trikentrin B (5), were isolated from the marine sponge Trikentrion flabelliforme . All possess antimicrobial activity and were identified by detailed spectroscopic analysis.

The complex of O-glucosylzeatin derivatives formed in Populus species☆

Abstract When zeatin was supplied to excised leaves of Populus alba , the principal metabolites formed were adenosine, O -β- d -glucopyranosyl- cis -zeatin (derived from cis -zeatin in the commercial zeatin used), O -β- d -glucopyranosylzeatin, and two new metabolites, namely, O -β- d -glucopyranosyldihydrozeatin and O -β- d -glycopyranosyl-9-β- d -ribofuranosyldihydrozeatin, the structures of which were confirmed by unambiguous synthesis. Chromatographic studies indicated that adenosine 5′-phosphate, zeatin 7-glucopyranoside, zeatin 9-glucopyranoside, dihydrozeatin and zeatin 9-riboside were minor metabolites. The principal metabolites of zeatin 9-riboside in P. nigra leaves were the new metabolites O -β- d -glucopyranosyl-9-β- d -ribofuranosylzeatin (synthesized chemically) and O -β- d -glucopyranosl-9-β- d -ribofuranosyldihydrozeatin.

Bacterial siderophores: the structures of the pyoverdins of Pseudomonas fluorescens ATCC 13525

Abstract The structures of five pyoverdins occurring in iron-deficient cultures of Pseudomonas fluorescens ATCC 13525 were elucidated using FAB-MS and 2D NMR techniques; they contain a common partly cyclic peptide containing a thirteen-membered ring bound to differently substituted chromophores derived from 2,3-diamino-6,7-dihydroxyquinoline.

Mild tagging procedures for the structural analysis of glycans

The reductive oxyamination of model glycan structures has been investigated as a mild, alternative tagging procedure to reductive amination using O-(4-nitrobenzyl)-hydroxylamine. Oxime formation was quantitative, but the reduction step did not always go to completion. Novel O- and N-substituted 7-hydroxycoumaryl- and 3-methoxybenzylhydroxylamines were synthesized and shown to couple quantitatively with model saccharides by oxime formation and reductive hydroxyamination, respectively, under very mild, aqueous conditions. The fluorescent derivatives produced show good chromatographic and mass spectrometric properties. Both procedures are suitable for the labeling of carbohydrates and oligosaccharide fragments from glycosaminoglycan structures, such as heparin and heparan sulfate.