Abstract
Marine cyanobacteria are a rich source of a variety of structurally-unique and biologically-active natural products. Isolation and structure elucidation of kalkitoxin, a potent neurotoxin from the marine cyanobacterium Lyngbya majuscula, is presented. Isolation of this compound was aided by bioassay-guided fractionation using the brine shrimp and gold fish toxicity assays. The planar structure of this thiazoline-containing lipid was elucidated by standard 1D and 2D NMR data. The stereochemistry of C-3 was determined to be R by Marfey's method, while the relative stereochemistry within the aliphatic chain of kalkitoxin was suggested to be 7R^*, 8S^*, 10S^* by J-based configuration analysis using a new NMR pulse sequence, HSQMBC, and a cryoproba NMR technology. Five synthetic isomers of kalkitoxin were compared to the natural compound by ^1H and ^ C NMR and CD spectroscopy. ^ C NMR chemical shifts showed very small differences in the range of less than 0.2ppm to natural kalkitoxin. However, the 3R,7R,8S,10S,2'R isomer showed maximal ^ C NMR differences of 0.026ppm with an average difference of only 0.008ppm. In addition, its CD spectrum was identical with natural kalkitoxin. Natural kalkitoxin is strongly ichthyotoxic (LC_ 700nM) and potently brine shrimp toxic (LC_ 170nM). Synthesized kalkitoxin was similarly potent in the brine shrimp assay (LC_ 170nM). Interestingly the synthesized enantiomer of kalkitoxin was relatively inactive (LC_ 9300nM). Kalkitoxin also showed potent NMDA receptor-mediated neurotoxicity (LC_ 3.86±1.91nM).