Two new karlotoxins found in Karlodinium veneficum (strain GM2) from the East China Sea

Abstract

The dinoflagellate Karlodinium veneficum is a harmful algal bloom species with a worldwide distribution. This small athecate dinoflagellate makes a family of polyketide toxins that are hemolytic, cytotoxic and ichthyotoxic. The first chemical structure for karlotoxins from East China Sea (ECS) is reported here. The two new karlotoxins, namely 4,5-dihydro-KmTx 2 (compound 1) and 4,5-dihydro-dechloro-KmTx 2 (compound 2), were isolated and purified from monoalgal cultures of K. veneficum strain GM2. Their structures were determined by spectroscopic analysis, including tandem mass spectrometry as well as 1D and 2D NMR experiments. These new karlotoxin congeners feature a saturated polyol arm different from previously reported for KmTx 2 that appears to increase hemolytic activity.

Keywords: Dinoflagellate; East China Sea; Hemolysis; Karlodinium veneficum; Karlotoxin.

Fig. 1

Chemical structures of compound 1 (4,5-dihydro-KmTx 2) and compound 2 (4,5-dihydro-dechloro-KmTx 2).

Fig. 2

Typical reverse phase HPLC chromatogram for the isolation of compound 1 (4,5-dihydro-KmTx 2) and compound 2 (4,5-dihydro-dechloro-KmTx 2). The UV spectra of both peaks are shown as inset.

Fig. 3

Structural elucidation by detailed analysis of 2D NMR data, including key HMBC (indicated by arrows from ¹H to ¹³C) and ¹H-¹H COSY (indicated by bold lines) for compound 1 and compound 2.

Fig. 4

(A) MS/MS spectrum of compound 1, which was similar with that of compound 2. (B) Elucidation of fragmentation pattern by tandem MS/MS of [M+Na]⁺. All m/z values correspond to ion fragments containing sodium ions.

Fig. 5

Comparative hemolytic potency for compound 1& compound 2 compared to KmTx 2 and saponin using Gilthead seabream (Sparus aurata) erythrocytes. Note the logarithmic scale for toxin amount. The fitted line is based on the Hill equation, and the HD50% estimates for these curves are presented in the legend to the figure.