Structure and absolute configuration of karlotoxin-2, an ichthyotoxin from the marine dinoflagellate Karlodinium veneficum

Abstract

In an attempt to determine the cause of repeated fish kills in an estuarine aquaculture facility in Maryland, a toxin with hemolytic, cytotoxic, and ichthyotoxic properties, designated as karlotoxin-2 (KmTx2), was isolated from Karlodinium veneficum. The structure of KmTx2 was elucidated by means of detailed ID and 2D NMR spectra, including 2D INADEQUATE. The relative and absolute configurations of KmTx2 were determined using J-based configuration analysis and comparison of its degradation products with synthetic controls.

Figure 1

Rotamers and coupling constants for C14∼C18

Figure 2

Correlation of relative configuration of C37∼C42 and coupling constant and NOE data

Figure 3

Degradation followed by esterification with MPA and Δδ^RS values

Figure 4

Histological sections (5-6 μm thickness) of zebrafish (Danio rerio) juveniles (Danio rerio) stained with hematoxylin and eosin (H&E) after exposure. (A) Control gill arch. (B) Karlotoxin-2 exposure (0.5 μg/ml for 1 hr) Note loss of intra-lamellar spacing and secondary lamellar fusion. (C) Gut epithelia of a striped killifish (Fundulus majalis) exposed in situ (∼0.2 μg/ml) during a fish kill event to karlotoxin.