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

Osama Mohamed,Zeinab Khalil,Robert Capon

doi:10.3390/md19030151

Osama Mohamed, Zeinab Khalil + Show 1 more

Open Access

https://doi.org/10.3390/md19030151

Copy DOI

Abstract

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).

Highlights

IntroductionA recent 2020 review used case studies in marine natural products chemistry to illustrate the heightened reactivity of selected natural products to environmental stimuli (i.e., pH, temperature, light, oxygen, metal ions, and solvents), where isolation, handling and/or storage promoted the formation of artifacts [1]
A recent 2020 review used case studies in marine natural products chemistry to illustrate the heightened reactivity of selected natural products to environmental stimuli, where isolation, handling and/or storage promoted the formation of artifacts [1]
One of the case studies used to illustrate this concept was that of the Schiff base prolinimines A–D (1–4) (Figure 1)

Summary

Introduction

A recent 2020 review used case studies in marine natural products chemistry to illustrate the heightened reactivity of selected natural products to environmental stimuli (i.e., pH, temperature, light, oxygen, metal ions, and solvents), where isolation, handling and/or storage promoted the formation of artifacts [1]. This review concluded, for a chemical to be viewed as a natural product it was not merely enough that it be isolated from a natural source, the compound should be detected in the fresh solvent extract prior to fractionation. It was noted that while standard isolation and handling techniques applied to CMB-F563 rice grain media cultivations yielded prolinimines B–D (2–4), high performance liquid chromatography (HPLC) analysis of fresh EtOAc extracts prior to any fractionation detected only prolinimines A–B (1–2). These observations prompted us to designate 1–2 as natural products, and 3–4 as artifacts. Consistent with this, we went on to document an in situ acid-mediated mechanism for the transformation of 1 to 3–4, informing a successful convergent biomimetic synthesis

Methods

Results

Discussion

Conclusion