Isolation and Structure Identification of Novel Brominated Diketopiperazines from Nocardia ignorata-A Lichen-Associated Actinobacterium

Abstract

Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of the actinobacterium Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted using an XAD7HP resin. Five known diketopiperazines: cyclo (l-Pro-l-OMet) (1), cyclo (l-Pro-l-Tyr) (2), cyclo (d-Pro-l-Tyr) (3), cyclo (l-Pro-l-Val) (4), cyclo (l-Pro-l-Leu) (5), and one auxin derivative: indole-carboxaldehyde (8) were isolated, along with two new brominated diketopiperazines: cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). Structure elucidation was performed using HRMS and 1D and 2D NMR analysis, and the synthesis of compounds 6 and 7 was carried out in order to confirm their structure.

Keywords: Nocardia; diketopiperazines; lichen-associated actinobacterium.

Figure 1

Structure of compounds 1–8 isolated from Nocardia ignorata.

Figure 2

Chemical profiling and isolation process of compounds 6 and 7. All samples were analyzed at 220 nm on Prevail reversed phase C₁₈ column with a gradient of H₂O (A)/acetonitrile (B) (10 min 100% of A, 30 min from 0% of B to 100% of B, 10 min 100% of B). Supernatant extract (SE) was fractionated using flash chromatography with a reversed phase to afford 14 fractions. Compounds 6 and 7 were isolated from the fifth fraction using semi-preparative HPLC on Prevail C₁₈ column. RE: resin extract.

Figure 3

HMBC and COSY correlations on compound 7.

Scheme 1

Synthesis of cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). TBTU: (O-(1H-Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), DIPEA: (N,N-Diisopropyléthylamine).