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

Abstract

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.