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Further investigation into a fish gut-derived fungus Evlachovaea sp. CMB-F563, previously reported to produce the unprecedented Schiff base prolinimines A-B ( 1 - 2 ), revealed a new cryptic natural product, N -amino-l-proline methyl ester ( 5 )-only the second reported natural occurrence of an N -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 5 to the Schiff base 9 , with the latter more readily detectable by UHPLC-DAD (400 nm) and HPLC-MS analyses. Moreover, we demonstrate that during cultivation 5 is retained in fungal mycelia, and it is only when solvent extraction disrupts mycelia that 5 is released to come in contact with the furans 7 - 8 (which are themselves produced by thermal transformation of carbohydrates during media autoclaving prior to fungal inoculation). Significantly, on contact, 5 undergoes a spontaneous condensation with 7 - 8 to yield the Schiff base prolinimines 1 - 2 , respectively. Observations made during this study prompted us to reflect on what it is to be a natural product (i.e., 5 ), versus an artifact (i.e., 1 - 2 ), versus a media component (i.e., 7 - 8 ).

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F-M ( 1 - 8 ) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure-activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F ( 1 ) was particularly noteworthy, with a 2.5 μM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).