High-resolution MS, MS/MS, and UV database of fungal secondary metabolites as a dereplication protocol for bioactive natural products

Abstract

A major problem in the discovery of new biologically active compounds from natural products is the reisolation of known compounds. Such reisolations waste time and resources, distracting chemists from more promising leads. To address this problem, dereplication strategies are needed that enable crude extracts to be screened for the presence of known compounds before isolation efforts are initiated. In a project to identify anticancer drug leads from filamentous fungi, a significant dereplication challenge arises, as the taxonomy of the source materials is rarely known, and, thus, the literature cannot be probed to identify likely known compounds. An ultraperformance liquid chromatography-photodiode array-high-resolution tandem mass spectrometric (UPLC-PDA-HRMS-MS/MS) method was developed for dereplication of fungal secondary metabolites in crude culture extracts. A database was constructed by recording HRMS and MS/MS spectra of fungal metabolites, utilizing both positive- and negative-ionization modes. Additional details, such as UV-absorption maxima and retention times, were also recorded. Small-scale cultures that showed cytotoxic activities were dereplicated before engaging in the scale-up or purification processes. Using these methods, approximately 50% of the cytotoxic extracts could be eliminated from further study after the confident identification of known compounds. The specific attributes of this dereplication methodology include a focus on bioactive secondary metabolites from fungi, the use of a 10 min chromatographic method, and the inclusion of both HRMS and MS/MS data.

Figure 1

Schematic of the workflow for the proposed dereplication protocol. Aliquots of active fungal extracts were evaluated using an UPLC-PDA-HRESIMS-MS/MS method, the data were analyzed using ACD Labs Intellixtract software and compared with an in-house database, and extracts producing known compounds were excluded.

Figure 2

A. (+)-ESI SIC of crude MSX 39480 extract (m/z: 681, 695, 697, 711, 725, 727, 741), B. Overlay chromatographic peaks of gliocladicillin C, C. (+)-HRESIMS of gliocladicillin C, and D. MS/MS CID fragmentation spectra of gliocladicillin C.

Figure 3

ACD/Intellixtract analysis of the TIC of an extract of MSX 39480.

Figure 4

(+)-ESI SIC of the crude extract of MSX 44407 (m/z: 668, 654, 640).

Figure 5

A. (+)-ESI TIC, B. SIC (m/z: 325, 355, 369, 378, and 397 for peaks 13, 11, 14, 12, and 15, respectively), and C. UPLC-PDA of the crude extract of MSX 40080.

Figure 6

(+)-HRESIMS and PDA spectra of compounds 11–15.