Analysis brevetoxin

Scheme 15. Retrosynthetic analysis of brevetoxin B (1) the first-generation approach.

This chapter deals with single crystal x-ray diffraction as a tool to study marine natural product structures. A brief introduction to the technique is given, and the structure determination of PbTX-1 (brevetoxin A), the most potent of the neurotoxic shellfish poisons produced by Ptychodiscus brevis in the Gulf of Mexico, is presented as an example. The absolute configuration of the brevetoxins is established via the single crystal x-ray diffraction analysis of a chiral 1,2-dioxolane derivative of PbTX-2 (brevetoxin B). 

Several brevetoxins have been examined for their respective abilities to competitively displace tritiated brevetoxin PbTx-3 from its specific site of action in brain synaptosomes. Analysis of IC q values revealed no marked differences in the displacing abilities between any of the type-1 toxins, and similarly there was no apparent difference between displacing abilities of PbTx-1 or -7, both type-2 toxins. Although some specific details require correlation, a gross comparison indicates that sodium channels in brain are similar in the systems examined. In the system studied most extensively, the rat brain synaptosome, t-test analysis revealed no significant differences between PbTx-2 and PbTx-3 IC q, or between PbTx-1 and PbTx-7 IC q, but statistically significant differences were found between the two classes (P<0.01) (5). If the Cheng-Prusoff equation (15) is applied ... 

Methods of detection, metabolism, and pathophysiology of the brevetoxins, PbTx-2 and PbTx-3, are summarized. Infrared spectroscopy and innovative chromatographic techniques were examined as methods for detection and structural analysis. Toxicokinetic and metabolic studies for in vivo and in vitro systems demonstrated hepatic metabolism and biliary excretion. An in vivo model of brevetoxin intoxication was developed in conscious tethered rats. Intravenous administration of toxin resulted in a precipitous decrease in body temperature and respiratory rate, as well as signs suggesting central nervous system involvement. A polyclonal antiserum against the brevetoxin polyether backbone was prepared a radioimmunoassay was developed with a sub-nanogram detection limit. This antiserum, when administered prophylactically, protected rats against the toxic effects of brevetoxin. 

The success of the soft ionization techniques (DCI, FAB, and LSIMS) presents several possibilities for detection of brevetoxins in complex matrices. Positive-ion DCI was used for the analysis of PbTx-3 metabolites generated in vitro by isolated rat hepatocytes (see below). Unmetabolized parent was conclusively identified and metabolites were tentatively identified, pending confirmation by alternate methods (see below). 

Brevetoxin A, synthesis, 258, 259 Brewer spectrophotometer, atmospheric analysis, 605... 

Scheme 2.6. Molecular structure and strategic bond disconnections (a), and retrosynthetic analysis (b, c) of brevetoxin A (35). Abbreviations for chemical groups Me, methyl Ph, phenyl Tr, trityl TBDPS, f-butyidiphenylsilyl ...

Rein, K.S., Baden, D.G., and Gawley, R.E. 1994. Conformational analysis of the sodium-channel modulator brevetoxin-A, comparison with brevetoxin-B conformations, and a hypothesis about the common pharmacophore of the site-5 toxins. Journal of Organic Chemistry 59, 2101-2106. 

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]). 

Brevetoxins administered in the form of [ H]-PbTx-3 distribute broadly to multiple tissues after intravenous, oral, and intratracheal administration to laboratory rats. Poll et al. first described the distribution of [ H]-PbTx-3 by intravenous administration to rats. Toxin distribution, reported as body burden, was nearly 70% to skeletal muscle and within 30 min of toxin administration 96% of the toxin distribnted to three tissnes skeletal muscle, liver, and intestine. HPLC analysis indicated that the PbTx-3 was intact in mnscle however, multiple products of metabolism were found in the feces. 

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