From a mutant strain of S. coccosporum, the new spirostaphylotrichins E (2), F (3), G (4 or 5), H (5 or 4), I (6), K (7), L (S), M (9), and S (10) have been isolated. Their structures have been elucidated by spectroscopic methods (UV, IR, 'H-NMR, "C-NMR, and MS), chemical transformations, and X-ray analysis (3 and 7).
Introduction. ~ The spirostaphylotrichins belong to a new family of microbial secondary metabolites whose first member, spirostaphylotrichin A (l), has been isolated by Peter and Auden from cultures of Stuphylotrichum coccosporum [l]. They possess a highly substituted spirocyclic system which contains a y -1actam moiety. In connection with biosynthetic studies [2], we have isolated additional spirostaphylotrichins from the wild type of S. coccosporum [3]. Having established acetate, a C,-dicarboxylic acid of the citric-acid cycle, most probably L-aspartate, and the methyl group of L-methionine as basic building blocks of 1, it was desirable to identify the more advanced precursors of the biogenetic pathway. Therefore, we have carried out a screening for mutants of S. coccosporum which were blocked in the production of 1, hoping to detect such intermediates. We now report on the isolation and structure elucidation of several new spirostaphylotrichins from the strain P84 of S. coccosporum.
Results. -Mutants of S. coccosporum were obtained by UV irradiation [4]. Spores were reported to occur in S.coccosporum, but we never had been able to detect them. Variation of the nutrition source, in particular the C-atom sources [5], strain maintenance on straw, and irradiation of cultures by light [6] never induced sporulation. Therefore, we had to use the mycelium for the UV irradiation. The mycelium was ground in a phosphate buffer with a mixer in the presence of quartz sand. The suspension was then irradiated with UV light and incubated on agar plates. After some days, new agar plates were inoculated by colonies from cells that had survived after the UV irradiation. After a period of 7 days, the colonies on these plates were examined for the presence of 1. Extracts of these colonies were analyzed by TLC [7] and compared with those of the wild type of S.coccosporum. By this procedure, 35 out of 635 strains (rate of surviving the UV irradiation was 0.2%) were obtained which did not produce any 1. In the extracts of the cultures of three out of these mutant strains (P84, P303, and P649), substances which differed from 1 were detected by TLC. Producing cultures of the mutant strain P303 yielded only very small amounts of crude extracts. The mutant strains P84 and P649 were subjected to further examination. The results obtained with P649 will be reported in a subsequent paper.