Brevetoxins structures

It was not until 1981 that the first brevetoxin structure was determined (13), PbTX-2, the most plentiful of the brevetoxins, was purified to crystallinity and the structure determined by x-ray crystallography. This important first step was the result of a three-group collatoration among Clardy, Lin, and Nakanishi. The yields of the major components from 50 L of culture (ca. 5 x 10 cells) were 0.8 mg of PbTX-1, 5.0 mg of PbTX-2, and 0.4 mg of PbTX-8. The structure of PbTX-2,... 

Scheme 14. Structure of brevetoxin B (1) and hypothetical polyepoxide precursors 71a and 71b.

Figure 3. The chemical structures of two classical alkaloid activators (batrachotoxin and veratridine) and of a recently characterized marine toxin [brevetoxin B (BvTX-B)], that acts at a different site on the Na channel.

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). 

Until 1986, the structure of PbTX-1, the most potent of the brevetoxins, remained unknown. Similarities in the and NMR spectra between PbTX-2... 

The crystal structures of PbTX-1 dimethyl acetal, PbTX-1, and dihydro PbTX-1 provide a total of four independent pictures of the same brevetoxin skeleton. It is rare that this quantity of structural data is available for a natural product of this size. A comparison of torsional angles shows that all four molecules have approximately the same conformations in all rings, except, of course, for the aldehyde side chain and the E-ring in one of the independent molecules of PbTX-1. Least squares superposition fits among the four molecules gave the following average distances ... 

Figure 1. The brevetoxins are based on two different backbone structures, as indicated (4). Type 1 toxins (top) include ...

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. 

Research in this area advanced in the 1970 s as several groups reported the isolation of potent toxins from P. brevis cell cultures (2-7). To date, the structures of at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). Early studies of toxic fractions indicated diverse pathophysiological effects in vivo as well as in a number of nerve and muscle tissue preparations (reviewed in 9-11). The site of action of two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds bind to a specific receptor site on the channel complex where they cause persistent activation, increased Na flux, and subsequent depolarization of excitable cells at resting... 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

These studies represent the first report of the metabolism of brevetoxins by mammalian systems. PbTx-3 was rapidly cleared from the bloodstream and distributed to the liver, muscle, and gastrointestinal tract. Studies with isolated perfused livers and isolated hepatocytes conflrmed the liver as a site of metabolism and biliary excretion as an important route of toxin elimination. [ H]PbTx-3 was metabolized to several compounds exhibiting increased polarity, one of which appeared to be an epoxide derivative. Whether this compound corresponds to PbTx-6 (the 27,28 epoxide of PbTx-2), to the corresponding epoxide of PbTx-3, or to another structure is unknown. The structures of these metabolites are currently under investigation. 

In other chapters of this volume considerable attention is given to marine toxins whose cellular sites of action have been identified. For example, saxitoxin, brevetoxin, and sea anemone toxins are prototypes of toxic molecules whose chemical structure is known, and whose actions on ionic channels in the cell membrane have been elucidated. Recent additions to such toxins are the piscivorus cone... 

---