Analysis palytoxin

The total synthesis of palytoxin (1) is a landmark scientific achievement. It not only extended the frontiers of target-oriented synthesis in terms of the size and complexity of the molecules, but also led to new discoveries and developments in the areas of synthetic methodology and conformational analysis. Among the most useful synthetic developments to emerge from this synthesis include the refinement of the NiCh/CrC -mediated coupling reaction between iodoolefins and aldehydes, the improvements and modifications of Suzuki s palladium-catalyzed diene synthesis, and the synthesis of A-acyl vinylogous ureas. 

Inhibition of EGF binding by palytoxin could be due to a decrease in receptor affinity, as in the case of TPA-type tumor promoters, and/or a decrease in receptor number. In Swiss 3T3 cells there are two classes of EGF receptors. The dissociation constants for the two EGF receptor classes were determined to be approximately 2 X 10 M and 2 x 10" M, corresponding to approximately 1 x 10 and 1 X 10 receptor molecules per cell, respectively (33). Scatchard analysis revealed that treatment of Swiss 3T3 cells with palytoxin, like PDBu, caused an apparent loss in high-affinity binding (Figure 2). However, in contrast to PDBu, palytoxin also caused a significant (approximately 50%) loss of low affinity EGF binding. 

Figure 2. Scatchard analysis of I-EGF binding to Swiss 3T3 cells treated with PDBu or palytoxin. Confluent quiescent Swiss 3T3 cells were treated at 37 C with solvent (o) or 200 nM PDBu ( ) for 15 min (Upper panel) or with solvent (o) or 11 pM palytoxin ( ) for 60 min (Lower panel). Cells were assayed as in Figure 1. (Reproduced with permission from Ref. 33. Copyright 1987 Cancer Research, Inc.)...

The extreme toxic potential of marine metabolites often prevents their application in medicine. However, a number of metabolites proved to be valuable tools in biochemistry, cell and molecular biology. For instance the neurotoxic maitotoxin [109-112] (interaction with extracellular calcium enhancement of calcium influx [113]), the neurotoxic brevetoxin B [114] (interaction with the binding-site-5 of voltage-sensitive sodium channels [115]), tetrodotoxin and saxitoxin (voltage clamp analysis to study sodium channels and excitatory phenomena [116] tetrodotoxin abolishes brevetoxin B activity [117]), okadaic acid [118-120] (analysis of phosphorylation and dephosphorylation processes in eukaryotic cell metabolism [121]), and palytoxin (stimulation of arachidonic acid metabolism synergistically with TPA-type promoters [122]). 

HPLC-DAD in comparison to reference palytoxin from P. toxica was carried out for further analysis of the BSE [27], The method employed a mobile phase of water acidified to pH 2.5 with trifluoro-acetic acid (solvent A) and pure acetonitrile (solvent B). A linear gradient was applied from 30% to 70% of solvent B over 45 min (see also Table 29.4). The BSF HPLC screening revealed two distinct peaks, which were eluted at approximately 38% acetonitrile with retention times very close to that of reference palytoxin. Both peaks showed the two characteristic UV absorption maxima, at 233 and 263 nm, while the ratio between their absorbance (233 versns 263 inn) was identical to that calculated for reference palytoxin. The peaks were collected separately and the toxic compounds purified from the BSF were called mascarenotoxin-A (McTx-A) and mascarenotoxin-B (McTx-B) [27],... 

Similar procedures are employed with regard to isolation and purification of palytoxin analogues derived from Ostreopsis spp. ceU cultures for subsequent toxicity studies or chemical analysis. 

Comparative Table of Most Important Quantitative Chemical Analysis Methods for Palytoxin and Analogues... 

Several chemical analysis methods have been developed for the determination of palytoxin and/ or palytoxin analogues, based on chemical properties characteristic and intrinsic to the toxin. Such methods include (1) infrared spectrometry, (2) ultraviolet spectrometry, (3) mass spectrometry, (4) high-performance capillary electrophoresis, (5) thin-layer chromatography, and (6) liquid chromatography. A comparative table of the most important quantitative chemical analysis methods summarizes the main features of each method and could assist in selection of the most fit-for-purpose analysis method depending on available equipment and sensitivity required (Table 29.4). 

The UV spectrum of palytoxin, as discussed earlier, shows two characteristic absorption peaks at 233 and 263 nm, contributed by the respective chromophores (Figure 29.4). The ratio of their absorbance (233 versus 263 mn), which is approximately 1.7 [3], is characteristic and indicative of the toxin s presence. The absorptions at either wavelength have been reported to be linearly related to palytoxin concentration in the range of 5-20 pg/mL. However, the disadvantage of this method is its detection limit, as the minimum detectable concentration has been reported to be 5 pg/mL (palytoxin standard in water), while toxicological and physiological effects have been observed with concentrations as low as 0.05-0.1 pg/mL [103], which limits its suitability as a regulatory analysis method. 

N acetic acid (1 1) flow rate 0.9 mL/min detection UV absorption at 263 nm]. A detection limit of 125 ng/injection for palytoxin was obtained by Mereish et al. [103] by use of a Bio-Sil 5 ODS column (Table 29.4) while Lau et al. [99] carried out the HPLC analysis of Caribbean palytoxin using a Novapak C18 reversed-phase column (75 mm x 3.9 mm), a gradient elution with 80% acetonitrile water (4 1) and detection at 230 nm. Reversed-phase HPLC was also applied for the analysis of Caribbean palytoxin [column Lichrospher 300 RP-8 mobile phase 0.1% TFA and 80% acetonitrile linear gradient detection UV absorption at 230 nm] with a lower tracing quantity of 10 [tg [21]. Other HPLC methods for palytoxin detection using different combinations of columns and solvent mixtures have also been reported [14,26]. 

MS on its own has been extensively used for identihcation and structure elucidation of palytoxin and/or its analognes. The observed low sensitivity of HPLC-UV methods with regard to palytoxin analysis, however, has triggered interest in the use of MS in combination with LC for the determination of these snbstances. Till date, there are four reports of LC-MS methods (Table 29.4) targeting palytoxin and its analognes. 

Riobo, R, Raz, B. and Franco, J.M., Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection. Analytica ChimicaActa, 566, 217, 2006. 

Mereish, K.A., Morris, S., Me Cullers, G., Taylor, T.J. and Bunner, D.L., Analysis of palytoxin by liquid chromatography and capillary electrophoresis. Journal of Liquid Chromatography, 14, 1025, 1991. 

Conformational analyses of low molecnlar weight bioactive componnds have given important information on their action mechanisms. X-Ray crystallographic analysis and NMR spectroscopy have served as the most nsefnl tools to obtain this information. However, apphcation of snch methods to palytoxin was confronted with great difficnlties dne to the noncrystaUine nature of the compound and heavy overlaps of signals in the NMR spectra. 

The chemistry of natural products encompasses their isolation, structure elucidation, partial and total synthesis, elucidation of their biogenesis, and the biomi-metic synthesis of N. p. Major breakthroughs in analysis were, e.g., the structural clarifications of morphine, lignin, insulin, estrones, and cholesterol as well as the elucidation of the biosyntheses of terpenoids, morphine, penicillin, chlorophyll, and vitamin B 2. Major advances in synthetic chemistry were, e.g., the total syntheses of camphor, hemin, quinine, saccharose, tropine, stryehnine, chlorophyll, vitamin B 2, erythromycin, taxol and palytoxin. Numerous N. p. of the so-ealled ehiral pool are used as starting materials for the synthesis of optically active compounds or serve (in the form of their derivatives) as catalysts for enantioselective syntheses. 

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