Application of New Technologies for the Rapid Identification of Compounds from Natural Sources
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
This thesis represents a continuation of the work on the isolation and structure elucidation of potential drug leads from terrestrial fungal sources that the natural products group at the University of Canterbury has been engaged in. Capillary NMR spectroscopy was involved in the research as the main tool for dereplication and elucidating the structures of novel bioactive compounds as well as for biosynthetic studies. Eleven new compounds including five cyclic peptides, four related pyrones and two diketopiperazines were isolated from the extract of Aspergillus sp. of endophyte collected from Malaysia. The five peptides F8268-A-1 to F8268-A-5 showed excellent P388 (HCT116 (ATCC CCL-247) and human breast cancer, MCF7 (ATCC HTB-22)) activities. Two of the peptides F8268-A-3 and F8268-A-5 were 4,000 times more active when compared with commercial drugs (fluorouracil, cisplatin and tamoxifen). The partial stereochemistries of F8268-A-2 and F8268-A-3 were established by Marfey’s method. Four related pyrones isolated from the same extract were also shown to have good P388 activities. They are related to the known compound NF00659A3. The relative stereochemistries were established from NOSEY experiments and the energy-minimised (MM2) model created using CHEM 3D software. Two new diketopiperazines, F7474-D3 and F7474-D11, also isolated from the Aspergillus extract did not show activity in the P388 assay. F7474-D11 contained the amino acid Me-kynurenine which is the first report of this from a natural source. The absolute stereochemistry of F7474-D11 was elucidated by Marfey’s method. The other diketopiperazine F7474-D3 was similar to the known compound lumpidin, and combined use of ROESY NMR and Marfey’s method established that the constituent amino acids had the unusual R configuration. Dereplication has been greatly improved by the application of capillary NMR. For example, the HPLC analysis and UV library searching of compounds from extracts F8095 and F7855 suggested they contained related compounds belonging to the lasiodiplodin family. However, CapNMR spectroscopic analysis and AntiMarin database searching revealed that the compounds from F8095 were all known polyesters while the compounds from F7855 did belong to the lasiodiplodin family. Two new lasiodiplodins were found in the F7855 extract, (3R,4R)-4-hydroxy-de-O-methyl-lasiodiplodin (F7855-4) and (E)-9-etheno-de-O-methyl-lasiodiplodin (F7855-6). The relative stereochemistries were elucidated from NMR coupling constant analyses. Two new dimers (F7090-A and F7090-B) were elucidated from a New Zealand fungal endophyte. The differences between these two dimers was their stereochemistries. F7090-A had the same stereochemistries for the three stereocentres in both parts, while the stereochemistry of F7090-B was different in the two parts of the dimer. Biosynthetic studies were also carried out using CapNMR methodology. A known compound tetrahydrofuran A and a new compound tetrahydrofuran B from an unidentified New Zealand fungus were used for this study. For the first time an INADEQUATE NMR experiment was successfully carried out using CapNMR spectroscopy, thus demonstrating the capability for carrying out biosynthetic studies on a very small scale (<200 μg of ¹³C-labelled compound). The implementation of efficient dereplication procedures with CapNMR methodology is paramount for those working in the field of natural products. The recent advances that have been made in the dereplication process in the natural products group at the University of Canterbury are given using examples from this research and where necessary from other group members.