Fervenulin

Derivatives of two new and unusual classes of heterocycles, possessing structural similarities to the broad spectrum antibiotic fervenulin, were synthesized and examined for in vitro antimicrobial activity. Only three of 17 mesoionic pyrimido[1,2-b]pyridazine-2,4-diones exhibited evidence of antimicrobial activity while seven of eight mesoionic pyridazino[2,3-a]-s-triazine-2,4-diones were active against one or more microorganisms. Susceptibility toward attack by nucleophiles of both mesoionic pyridazino[2,3-a]-s-triazine-2,4-diones and fervenulin was observed.

Toxoflavin (1), fervenulin (2), and reumycin (3), known to be produced by plant pathogen Burkholderia glumae BGR1, are structurally related 7-azapteridine antibiotics. Previous biosynthetic studies revealed that N-methyltransferase ToxA from B. glumae BGR1 catalyzed the sequential methylation at N6 and N1 in pyrimido[5,4-e]-as-triazine-5,7(6H,8H)-dione (4) to generate 1. However, the N8 methylation of 4 in the biosynthesis of fervenulin remains unclear. To explore the N-methyltransferases required for the biosynthesis of 1 and 2, we identified and characterized the fervenulin and toxoflavin biosynthetic gene clusters in S. hiroshimensis ATCC53615. On the basis of the structures of intermediates accumulated from the four N-methyltransferase gene inactivation mutants and systematic enzymatic methylation reactions, the tailoring steps for the methylation order in the biosynthesis of 1 and 2 were proposed. The N-methylation order and routes for the biosynthesis of fervenulin and toxoflavin in S. hiroshimensis are more complex and represent an obvious departure from those in B. glumae BGR1.

The structures of xanthothricin (I) and fervenulin (II), pyrimido-[5,4-e]-1,2,4-triazine antibiotics were determined by x-ray analysis. The crystals of I and II are monoclinic: a 9.667(6), 18.270(7), b 14.029(9), 5.723(2), c 6.914(3), 18.058(7) A, beta 116.47(3), 118.90(2) degrees, Z 4,8; space group P21/c. Analysis of bond length distribution in I and II was indicative of significant alternation of the double and ordinary bonds.