-Amino-l-Proline Methyl Ester from an Australian Fish Gut-Derived Fungus: Challenging the Distinction between Natural Product and Artifact.

Further investigation into a fish gut-derived fungus sp. CMB-F563, previously reported to produce the unprecedented Schiff base prolinimines A-B (-), revealed a new cryptic natural product, -amino-l-proline methyl ester ()-only the second reported natural occurrence of an -amino-proline, and the first from a microbial source. To enable these investigations, we developed a highly sensitive analytical derivitization methodology, using 2,4-dinitrobenzaldehyde (2,4-DNB) to cause a rapid in situ transformation of to the Schiff base , with the latter more readily detectable by UHPLC-DAD (400 nm) and HPLC-MS analyses. Moreover, we demonstrate that during cultivation is retained in fungal mycelia, and it is only when solvent extraction disrupts mycelia that is released to come in contact with the furans - (which are themselves produced by thermal transformation of carbohydrates during media autoclaving prior to fungal inoculation). Significantly, on contact, undergoes a spontaneous condensation with - to yield the Schiff base prolinimines -, respectively. Observations made during this study prompted us to reflect on what it is to be a natural product (i.e., ), versus an artifact (i.e., -), versus a media component (i.e., -).