Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214

Abstract

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A–D (11–14) in addition to a suite of very minor aza analogues 1–6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1–6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N–P (7–9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1–15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1–5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2′ substituted analogues 3–5.

Chrysosporazines Revisited: Regioisomeric Phenylpropanoid Piperazine P-Glycoprotein Inhibitors from Australian Marine Fish-Derived Fungi

“…Based on our earlier investigations into azachrysosporazines from Chrysosporium sp. CMB-F214 [ 7 ], we speculated that 3 – 4 differed from 1 – 2 by the incorporation of a nicotinamide rather than a benzamide moiety, and that supplementation of CMB-F661 cultivations with sodium nicotinate would enhance production levels to the point where isolation, characterisation and structure elucidation was feasible. As predicted, comparable fractionation of PDA cultivations of CMB-F661 supplemented with sodium nicotinate yielded two new minor azachrysosporazines 3 – 4 .…”

“…Indeed, chrysosporazine B exhibited an impressive 3.2- fold increase (FI) in inhibitory potency compared to the positive control verapamil, as measured by the ratio of the respective gain in sensitivity (GS) values, where GS is the ratio of increased sensitivity (IC 50 ) of SW600 Ad300 cells treated with doxorubicin, with versus without analyte supplementation (i.e., 2.5 μM chrysosporazine B or verapamil). Buoyed by this discovery, and keen to further advance the structure activity relationship (SAR) investigations, in 2021 we reported a successful detection, production, isolation, characterisation, identification and evaluation of the P-gp inhibitory properties of an array of new chrysosporazines, including minor azachrysosporazines [ 7 ], and followed this with a 2022 account of precursor directed unnatural neochrysosporazines [ 8 ]. These latter two investigations were assisted by; (i) cultivation profiling using a miniaturized 24-well plate microbioreactor format (known in lab as the MATRIX), which allowed for solid as well as static and shaken broth culture formats, and a wide array of media compositions, including the addition of natural and unnatural biosynthetic precursors; (ii) a rapid UPLC-QTOF-MS/MS and global natural products social (GNPS) molecular networking analysis to rapidly detect and prioritize new from known chrysosporazines in complex extracts and fractions; (iii) precursor directed biosynthesis using cultivation supplementation with natural substrates to amplify the production of exceptionally minor natural aza analogues (i.e., azachrysosporazines); and (iv) precursor directed biosynthesis using cultivation supplementation with unnatural substrates to repurpose the biosynthetic machinery to produce new analogues (i.e., neochrysosporazines).…”

“…CMB-F214 and CMB-F294 to produce a wide array of structurally diverse chrysosporazines. For example, solid phase M1 cultures of CMB-F214 supplemented with sodium nicotinate upregulated and facilitated access to the minor co-metabolite azachrysosporazines A1–A2, B1, C1–C2 and D1, chrysosporazines N–P and spirochrysosporazine [ 7 ], while supplementation with sodium picolinate yielded neochrysosporazines A–E, and with sodium isonicotinate yielded neochrysosporazines F–J [ 8 ]. By contrast, solid phase M2 cultures of CMB-F214 without precursor supplementation yielded the new chrysosporazine Q [ 7 ].…”

“…For example, solid phase M1 cultures of CMB-F214 supplemented with sodium nicotinate upregulated and facilitated access to the minor co-metabolite azachrysosporazines A1–A2, B1, C1–C2 and D1, chrysosporazines N–P and spirochrysosporazine [ 7 ], while supplementation with sodium picolinate yielded neochrysosporazines A–E, and with sodium isonicotinate yielded neochrysosporazines F–J [ 8 ]. By contrast, solid phase M2 cultures of CMB-F214 without precursor supplementation yielded the new chrysosporazine Q [ 7 ]. Building on this, solid phase YES cultures of CMB-F294 supplemented with sodium coumarate yielded the new neochrysosporazines K–L and chrysosporazines R–S, while sodium ferulate yielded the new neochrysosporazines M–O, and sodium benzoate yielded the new chrysosporazines R–S, neochrysosporazines P–Q [ 8 ], and the previously reported hancockiamide C [ 9 ].…”

“…In this context, the chrysosporazine and azachrysosporazine trivial nomenclature refers to biosynthetically-related natural products , and neochrysosporazine refers to precursor-derived unnatural products . To guide discussion we apply the collective term chrysosporazines to encompass all known members of this extended structure class, including chrysosporazines [ 5 , 6 ], azachrysosporazines [ 7 ], and spirochrysosporazine [ 7 ] and neochrysosporazines [ 8 ] ( Figure 1 ), as well as the biosynthetically related hancockiamides [ 9 ], brasiliamides [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ], iizukine E [ 19 ] and helvamide [ 20 ] ( Figure 2 ).…”