Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae.

The estuarine genus Pfiesteria has received considerable attention since it was first identified and proposed to be the causative agent of fish kills along the mid-Atlantic coast in 1992. The presumption has been that the mechanism of fish death is by release of one or more toxins by the dinoflagellate. In this report, we challenge the notion that Pfiesteria species produce ichthyotoxins. Specifically, we show that (i) simple centrifugation, with and without ultrasonication, is sufficient to "detoxify" water of actively fish-killing cultures of Pfiesteria shumwayae, (ii) organic extracts of lyophilized cultures are not toxic to fish, (iii) degenerate primers that amplify PKS genes from several polyketide-producing dinoflagellates failed to yield a product with P. shumwayae DNA or cDNA, and (iv) degenerate primers for NRPS genes failed to amplify any NRPS genes but (unexpectedly) yielded a band (among several) that corresponded to known or putative PKSs and fatty acid synthases. We conclude that P. shumwayae is able to kill fish by means other than releasing a toxin into bulk water. Alternative explanations of the effects attributed to Pfiesteria are suggested.

Computational, ReactIR-, and NMR-spectroscopic investigations on the chiral formyl anion equivalent N-(alpha-lithiomethylthiomethyl)-4-isopropyl-5,5-diphenyloxazolidin-2-one and related compounds.

The 4-isopropyl-3-methylthiomethyl-5,5-diphenyloxazolidin-2-one is readily lithiated in THF on the exocyclic CH2 group (1 --> 2) to give a synthetically useful chiral nucleophilic formylating reagent. We have now studied the lithiation reaction by ReactIR spectroscopy and the structure of the organolithium reagent by computational methods and by NMR-spectroscopic measurements. The lithiation is complete at -78 degrees C within 90 seconds, and it is accompanied by a decrease of the C=O wavenumber by 50 cm(-1). The NMR data (collected in [D8]THF) give no evidence for 13C,6Li coupling or for aggregation; from DPFGSE-ROE spectra the single diastereoisomer of the lithium compound 2 seen in the NMR spectra (NMR-spectroscopic measurements from -105 to -20 degrees C) is assigned like configuration; the 13C=O signal of the oxazolidinone undergoes a 7 ppm downfield shift upon lithiation (1 --> 2); line-shape analyses of the signals from the diastereotopic CH2 and CMe2 protons in lithiated 4,4-dimethyl-3-methylthiomethyloxazolidin-2-one (model compound 7) reveal a deltaH(double dagger) of 8.9 +/- 0.2 kcal mol(-1) for enantiomerization. The theoretical calculations provide an energy-minimum structure for the lithium compound 2 with coordination of the carbonyl oxygen to lithium, with an antiperiplanar arrangement of the C,Li and S,CH3 bonds, and with relative like configuration of the two stereocenters--in perfect agreement with the conclusions from the IR- and NMR-spectroscopic measurements!

Synthesis and reactivity of dipole-stabilized but unchelated alpha-aminoorganolithiums.

Two N-methyl-5-lithio-2-pyrrolidinones have been prepared by tin-lithium exchange. These two alpha-aminoorganolithium compounds that are stabilized by an amide dipole, but not by chelation to the amide carbonyl. Both constitute test cases for comparing the stability and reactivity of "dipole-stabilized" and "unstabilized" alpha-aminoorganolithiums. We find that active methylene protons interfere with the reaction, so geminal disubstitution alpha to the amide carbonyl was necessary to suppress side reactions. These species do not react as efficiently as unstabilized alpha-aminoorganolithiums, or even as well as chelated dipole-stabilized alpha-aminoorganolithiums, toward typical electrophiles. The tin-lithium exchange to form these species was also not as facile as with other alpha-aminoorganostannanes.

Synthesis and reactivity of conformationally locked alpha-aminoorganostannanes and alpha-aminoorganolithiums. Discovery of a surprising configurational requirement for transmetalation.

[equation--see text] 2-Tributylstannyl-N-methylpiperidines that are conformationally locked by a 4-tert-butyl substituent were evaluated in transmetalations (Sn-Li exchange) and reactions with electrophiles. When the tin is equatorial, transmetalation occurs smoothly as does reaction with carbonyl electrophiles. Alkyl halides seem to undergo single electron transfer reactions, affording nonselective alkylation products, along with products of radical disproportionation. In a surprise, an axially oriented tin failed to transmetalate, suggesting that a synclinal relationship between the nitrogen lone pair and the carbon-tin bond is a requirement for transmetalation.

Unusual conformational effect in alpha-aminoorganostannanes.

[formula: see text] Dynamic NMR analysis of conformationally mobile and rigid 2-tributylstannyl-N-methylpiperidines revealed an unexpected conformational effect that is manifested in a small energy difference between conformers in which the tin is equatorial and axial. The major reason appears to be a distortion of the conformer in which the C-2-Sn bond is synclinal to the nitrogen lone pair.

The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels.

BACKGROUND: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

Is the A-ring lactone of brevetoxin PbTx-3 required for sodium channel orphan receptor binding and activity?

Brevetoxin PbTx-3 and non-toxic derivative 4 were investigated for their abilities to bind to the specific brevetoxin receptor site on rat brain synaptosomes and to modulate the normal function of voltage-gated sodium channels as determined by patch clamping of cultured neurons. Compounds 4 and 5 are produced from PbTx-3 by opening of the A-ring lactone to the saturated and unsaturated diols using sodium borohydride in ethanol. Natural PbTx-3 exhibited tighter binding to rat brain synaptosomes by at least 3 orders of magnitude as determined by competitive radioligand binding experiments, and was also more effective at activating voltage-gated channels. Patch clamping revealed the 3 orders of magnitude greater potency of PbTx-3 toxin over 5, although each produced delayed sodium channel opening and a pronounced delay in inactivation. Conformational modeling of the Brevetoxin B backbone indicates that the two molecules are identical except for the region of the A-Ring lactone. Thus, we conclude that the brevetoxin PbTx-3 backbone requires electrophilic functionality in the region of the lactone in PbTx-3, and that opening of the ring in 5 is sufficient to substantially reduce both binding and activity.

Binding of brevetoxins and ciguatoxin to the voltage-sensitive sodium channel and conformational analysis of brevetoxin B.

The marine toxins known generically as brevetoxins, as well as their structural relative ciguatoxin, are known as polyether ladder toxins, and bind uniquely to site 5 of the voltage-sensitive sodium channel. Rat brain synaptosome binding data show similarities in binding affinity for brevetoxins having the same structural (ladder) backbone, but different affinities between brevetoxins having different backbones. Ciguatoxin has a different backbone from the brevetoxins, but binds even more strongly to the same site. Could the flexibility of the backbone be related to their relative toxicities? As part of an effort to identify the common pharmacophore for the toxins, Monte Carlo methods were used to generate conformational models of the polyether ladder toxin brevetoxin B (PbTx-2) which shows significant flexibility at the juncture of the two 7-membered rings.