Homolog separation, a necessity for the proper identification of fungal metabolites.

Monitoring of fungal extracts for the production of novel metabolites, using a modular analytical system combining HPLC with UV-MS-ELS detection, identified culture LL-W1278 as a fungus producing new biopolymers. Only a non-routine HPLC analysis of a culture extract revealed that the standard water-acetonitrile elution method did not separate all members of the metabolite complex. Fine-tuning the eluting solvents established that it was essential to include acid with the water-methanol system to separate the new materials. The routinely used water-acetonitrile system, with or without acid, was incapable of separating all homologues. With the modified method the new homologues W1278-Ax, Bx, and Cx were separated. LC/MS analysis indicated that these compounds had molecular weights of 706, 900, and 1094, respectively, 44 mass units lower than their three major homologues, W1278-A, B, and C, identified previously. UV and NMR data as well as mass fragmentation patterns established unambiguously that the new compounds lacked a carboxyl group at the terminal resorcinol unit of the biopolymer, consisting of several catenated hydroxymellein residues. A time study concerning the stability of these fungal metabolites showed a slow, but complete degradation of the primary metabolites over several months when kept as a DMSO solution.