Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Curaçao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.

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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...