Biosynthesis and transformation of homoanatoxin-a in the cyanobacterium Raphidiopsis mediterranea skuja and structures of three new homologues

The biosynthetic origin of the C-12 methyl group in homoanatoxin-a (1) was identified by the feeding experiment of L-[methyl-¹³C]-methionine in the culture of the cyanobacterium Raphidiopsis mediterranea Skuja strain LBRI 48. Remarkably high incorporation (80%) of ¹³C was observed at C-12. The in vivo enzymatic transformation of 1 was also examined by the prolonged culture of strain LBRI 48. The cells harvested at the stationary phase (15 days of incubation) gave higher contents of 4S-hydroxyhomoanatoxin-a (2), 4R-hydroxyhomoanatoxin-a (3), 2,3-epoxyhomoanatoxin-a (4), and 4-ketohomoanatoxin-a (5) than those from the cells collected at the late logarithmic growth phase (5 days). Compounds 2-5 would be transformed from 1 in the cells. The ratio of anatoxin-a and 1 was not significantly changed between two phases. Compound 5 was generated from 1 by air oxidation during storage even under dry and cool (-30 °C) conditions, but the oxidation was prevented in a water solution at both room temperature and -30 °C (frozen stock). Homoanatoxin-a (1) gave 2,3-dihydro-3-methoxyhomoanatoxin-a (6) during the separation procedures probably by the Michael reaction of methanol used as solvent. It should be noted that 4 was isolated for the first time from a cyanobacterium as the natural product. Compounds 3 and 5 were new members of the anatoxins.