Structure, Synthesis, and Biological Properties of Kalkitoxin, a Novel Neurotoxin from the Marine Cyanobacterium Lyngbya majuscula

Abstract

Cyanobacteria from a diversity of marine and freshwater habitats are known to produce neurotoxic secondary metabolites. 1 Herein, we describe the complete stereostructure, synthesis, and biological properties of kalkitoxin (1), a novel neurotoxic lipopeptide from a Caribbean collection of Lyngbya majuscula.The organic extract of this L. majuscula exhibited potent brine shrimp and fish toxicity. 2 Using these assays, the toxic metabolite kalkitoxin (1), was isolated by sequential silica gel VLC, CC, and normal-phase HPLC (12.8 mg, 0.3% of extract). Subsequently, bioassay-guided fractionation using a primary cell culture of rat neurons in a microphysiometer 3 or inhibition of IL-1 stimulation of sPLA 2 in hepatocarcinoma cells 4 led to re-isolation of 1 in small yield from various Caribbean collections of L. majuscula.Kalkitoxin (1) analyzed for C 21 H 38 N 2 OS indicated four degrees of unsaturation; from 13 C NMR analysis in DMSO-d 6 two were due to double bonds, one to a carbonyl group, and the remaining one to a ring system. 5 Data from E.COSY, HSQC, and a modified HSQMBC 6 experiments in benzene-d 6 allowed deduction of six partial structures for 1 (Supporting Information). One partial structure was composed of a sec-butyl group in which the methine component was deshielded to a chemical shift (δ 2.28) consistent with its being adjacent to a carbonyl. A second partial structure was composed of a methylated tertiary amide group which existed in two conformations (Supporting Information). A third partial structure possessed a deshielded methylene (δ 3.35) that could be sequentially connected by E.COSY to a second methylene group, and by HSQMBC to a methine and high-field methyl group. By E.COSY, an additional high-field methylene group (δ 1.10, 1.02) was adjacent to a methine which also bore a methyl group. The fifth partial structure was composed of a similar -CH 2 -CH-CH 3 grouping; however, in this case, the methylene group protons were deshielded to δ2 .31 and δ 2.55. The final partial structure, based on E.COSY correlations, HSQMBC, and chemical shift models, 7 was composed of a thiazoline ring with an ethylene appendage; this was further substantiated by EIMS fragmentations (Supporting Information). HSQMBC data were used to connect these partial structures and gave the full planar structural assignment of 1.The C3 stereochemistry of kalkitoxin was determined by Marfey's analysis. Kalkitoxin was ozonized and then hydrolyzed in 6 N HCl to obtain cysteic acid. Marfey's analysis of this hydrolysate yielded L-cysteic acid, defining C3 as R. The limited amount of kalkitoxin precluded determination of the C2′ stereochemistry. The relative stereochemistry of the three chiral centers within the aliphatic chain of kalkitoxin (C7, C8, C10) was determined using the J-based configuration analysis method. 8 The 3 J CH values were measured by a modification of the recently reported HSQMBC pulse sequence, 6 and the 3 J HH values were determined utilizing the E.COSY pulse sequence. 9 To overcome the limited sample size...

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