Kathleen S. Rein

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

BACKGROUNDnBrevetoxins 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.nnnRESULTSnSeveral 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.nnnCONCLUSIONSnOur 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.

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.

Single electron transfer in the addition of chiral dipole-stabilized organolithiums to carbonyls. Stereochemistry of a chiral nucleophile as a mechanistic probe

Abstract Spectroscopic and stereochemical evidence is provided which suggests that single electron transfer (SET) accounts for the difference in selectivity observed in the reaction of metalated pivalamides and oxazolines with carbonyls.

Brevetoxin-3: Total assignment of the 1H and 13C NMR spectra at the submicromole level

Abstract The proton and carbon NMR spectra of the marine polyether toxin, brevetoxin-3, are totally assigned using a series of 2D NMR experiments which included: TOCSY, ROESY, HMQC, HMBC, and IDR-(Inverted Direct Response)-HMQC-TOCSY. All work was performed on a sample consisting of 800 μg (0.95 μmole) at 500 MHz.

Synthesis of (−)-egenine (decumbensine) by asymmetric carbonyl addition

Abstract The first synthesis of the phthalideisoquinoline hemiacetal egenine is reported, along with spectral data suggesting that egenine and decumbensine are one and the same. These synthetic studies are part of a larger investigation into the face-selectivity of additions of chiral dipole-stabilized organometallics to aldehydes. The synthesis of egenine and the correlation to bicuculline diol establish the absolute configuration of the major stereoisomer formed as being opposite to that expected based on earlier work.

Brevetoxin PbTx-2 immunology: differential epitope recognition by antibodies from two goats.

The epitopic regions of the brevetoxin PbTx-3 molecule, produced by the marine dinoflagellate Ptychodiscus brevis, have been identified by structural modification at three distinct regions of the toxin. These are: the A-ring lactone region of the molecule, the K-ring side-chain and the H-ring. The modified PbTx-3 derivatives were tested for their ability to bind brevetoxin goat antisera directed against the PbTx-3 molecule, by radioimmunoassay. The results showed that at least two major epitopes and one minor epitope are recognized: the A-ring lactone region of the molecule and the K-ring side-chain, and the H-ring. The results illustrate the variety of antibodies which may be produced, even within a species, and suggests that epitope characterization is important in the development of assays which are to be employed in seafood safety issues.

Biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid moiety and absolute configuration of pahayokolides A and B.

Pahayokolides A and B are cyclic undecapeptides that were isolated from the cyanobacterium Lyngbya sp. They contain the unusual α-hydroxy-β-amino acid 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configurations of the amino acids of the pahayokolides, except for the four oxygen-bearing stereocenters of Athmu, have been determined by Marphys method. Incorporation of labeled leucine and acetate precursors into the pahayokolides has established that Athmu is derived from a leucine or α-keto isocaproic acid starter unit, which is further extended with three acetate units.

Marine Toxins: How They are Studied and What They Can Tell Us

Throughout the world, marine toxins are responsible for a variety of diseases in humans (Table 26.1; Baden et al., 1995), as well as maladies in other mammals, fish and birds. The diseases in humans range from acute neurologic diseases, such as ciguatera and paralytic shellfish poisoning, to chronic dementia as reported with domoic acid exposure. Most of the toxins are small, non-peptidic materials that are highly potent; exceedingly small quantities can lead to illness (i.e. <1 mg/kg body weight).

Alkylations of Nitrogen-stabilized Carbanions

The deprotonation of an sp3-hydrogen α to nitrogen has been developed into a highly useful synthetic tool in recent years.1,2 Although some authors have suggested that this type of reactivity is a reversal of the ‘normal’ reactivity adjacent to nitrogen,2,3 we submit that this notion is inappropriate and should be discontinued. The α-deprotonation of dimethyldodecylamine4 and triethylamine5 was reported over 20 years ago. In 1984, Ahlbrecht reported that s-butylpotassium readily deprotonates N-methylpiperidine, N-methylpyrrolidine and triethylamine, and that the derived organometallics add readily to aldehydes, ketones and alkyl halides.6 An example is shown in Scheme 1.

2.1 – Nitrogen Stabilization

This chapter covers the carbonyl addition chemistry of carbanions stabilized by a nitrogen atom or a nitrogen-containing functional group in which the nitrogen is responsible for the stabilization. In most cases, the carbanions are formed by deprotonation, but metal–halogen exchange is occasionally important. A carbanion that is stabilized by a nitrogen may exist in three oxidation states: sp, sp2 or sp3. The simplest nitrogen-stabilized carbanion is cyanide, the sp-hybridized case. In recent years, most efforts in this area have been expended on developing the chemistry of sp2- and sp3-carbanions. This chapter deals with the addition of these types of anions to carbonyl compounds. Alkylation reactions of sp3-hybridized species are covered in Volume 3, Chapter 1.2, and alkylation of sp2-hybridized carbanions is covered in Volume 3, Chapter 1.4. Specifically excluded from this chapter are additions of carbanions stabilized by a nitro group (the Henry nitroaldol reaction) and azaenolates, which are covered in Volume 2, Chapters 1.10, 1.16 and 1.17.