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...

M arine cyanobacteria represent a particularly rich source of structurally unique neurotoxic secondary metabolites (1-5). Lyngbya majuscula is a pantropical marine cyanobacterium that is the source of antillatoxin (ATX), a structurally unusual lipopeptide (1) (Fig. 1). Blooms of L. majuscula have been associated with adverse effects on human health. These blooms have been reported to cause respiratory irritation, eye inflammation, and severe contact dermatitis in exposed fishermen and swimmers (6). ATX has been shown to be among the most ichthyotoxic metabolites isolated to date from a marine microalga (1) and, more recently, has been demonstrated to be neurotoxic in primary cultures of rat cerebellar granule cells (4). In the latter study, morphologic evidence of ATX-induced neurotoxicity included swelling of neuronal somata, thinning of neurites, and blebbing of neurite membranes. ATX also induced a concentration-dependent cytotoxicity in cerebellar granule neurons as monitored by lactate dehydrogenase efflux (ATX EC 50 ϭ 20.1 Ϯ 6.4 nM) (4). This neurotoxic response of ATX was prevented by coexposure with noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor such as MK-801 and dextrorphan.Voltage-gated sodium channels are responsible for generation of the rising phase of the action potential in membranes of neurons as well as in most other electrically excitable cells. Sodium channels consist of a pore-forming ␣ subunit of 260 kDa associated with auxiliary ␤ subunits of 33-36 kDa (7,8). Voltagegated sodium channels represent the molecular target for an array of natural products including marine neurotoxins. Marine neurotoxins such as tetrodotoxin (TTX), saxitoxin, conotoxins, sea anemone toxins, brevetoxin, and ciguatoxin all bind with high affinity and specificity to at least six distinct receptor sites on sodium channel ␣ subunits (8). Collectively, these toxins have served as important tools to explore the structure and function of voltage-dependent sodium channels. These marine neurotoxins produce characteristic alterations in the two major properties of sodium channels, namely ion permeation and gating (8).The objective of the present study was to test the hypothesis that ATX acts as an activator of voltage-dependent sodium channels. This hypothesis emanated from our previous studies which were consistent with ATX acting as an autocrine excitotoxic agent in cerebellar granule neurons. In this study we have used a combination of neurochemical and pharmacological approaches to show that ATX is a structurally unusual lipopeptide activator of neuronal voltage-gated sodium channels. Materials and MethodsMaterials. Tritiated batrachotoxin A 20-␣-benzoate ([ 3 H]BTX) and 22 Na ϩ were obtained from DuPont͞NEN. Deltamethrin was purchased from Biomol (Plymouth Meeting, PA). Veratridine, brevetoxin-1 (PbTx-1), and ouabain were obtained from Sigma. Sea anemone toxin was purchased from Calbiochem. Fluo-3 AM and pluronic acid were obtained from Molecular Probes. ATX was either authentic natural (Ϫ)-ant...