Elsevier

Tetrahedron

Volume 58, Issue 33, 12 August 2002, Pages 6655-6658
Tetrahedron

Biosynthetic studies of the tetramic acid antibiotic trichosetin

https://doi.org/10.1016/S0040-4020(02)00689-0Get rights and content

Abstract

A feeding experiment with 13C-labeled precursors was conducted in the dual cultures of Trichoderma harzianum H14 and Catharanthus roseus callus to determine the biosynthetic origin of the carbon atoms in trichosetin. Results showed that trichosetin originated from two separate biogenetic units. One unit is an octaketide intermediate directly derived from eight intact acetate units joined in a head-to-tail fashion of a polyketide pathway. The other unit is serine.

Incorporation of 13C labeled precursors revealed that trichosetin originated from an octaketide intermediate and serine.

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Introduction

Trichoderma harzianum H14, when co-cultured with Catharanthus roseus callus on a hormone-free Murashige and Skoog medium, has been shown to produce trichosetin.1 Trichosetin (1) exhibits a remarkable antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and Bacillus subtilis. Structural elucidation revealed that it is a N-desmethyl homolog of equisetin (2), an antibiotic first isolated from Fusarium equiseti.2 Another related compound is phomasetin (3) isolated from Phoma sp.3 Equisetin and phomasetin are also toxic to Gram-positive bacteria and both of them have anti-HIV integrase activity.

Aside from their diverse biological activities, investigation of this series of fungal metabolites is very interesting because they are consisted of two distinctive structural features. The first feature is the 2,4-pyrrolidinedione moiety which is the characteristic structural element of all tetramic acid antibiotics. In tenuazonic acid, erythroskyrine, malonomicin, streptolydigin and aflastatin A, C-2 and C-3 of this moiety were found to be derived from acetate, whereas C-4 and C-5 were from a certain amino acid, i.e. isoleucine, valine, serine, glutamate and alanine, respectively.4., 5., 6., 7., 8. Carbons 4′, 5′ and 6′ of the analogous structure in equisetin and phomasetin were also proposed to originate from serine3 based on the utility of this amino acid in the total synthesis of equisetin.9 The second structural feature of interest is the bicyclic hydrocarbon moiety. The presence of a similar feature occurs in ilicicolin H where it was found to be derived through cyclization of a linear polyketide.10

In order to understand better the details of the formation of these two structural moieties, we investigated the biosynthesis of trichosetin by growing the dual culture of T. harzianum and C. roseus callus with stable isotope-labeled precursors. Labeled trichosetin was then isolated and the labeling pattern analyzed by 13C NMR spectroscopy. This paper is the first report dealing with the biosynthesis of trichosetin.

Section snippets

Results and discussion

In order to elucidate the biosynthetic origin of the carbon atoms in trichosetin, the dual culture of T. harzianum and C. roseus callus was grown in a solid medium supplemented with [1-13C], [2-13C] or [1,2-13C2] acetate. Labeled trichosetin was purified and the 13C NMR spectra were then analyzed by comparison with the 13C NMR spectrum of the unlabeled trichosetin. Chemical shift assignments were as previously reported,1 as shown in Table 1. Trichosetin isolated from cultures supplemented with

General

13C NMR spectra were recorded on a JEOL 400 spectrometer in CD3OD (99.8% atom enriched, Cambridge Isotope Laboratories, Inc., USA) at −80°C. The residual solvent signal (δC=49.9 ppm) was used as internal standard. All isotopically labeled compounds were 99% atom enriched and purchased from Isotec Co. (OH, USA).

Culture conditions

Isotopically labeled compounds were dissolved in water, filter-sterilized, and added into a hormone-free MS medium14 containing 1% sucrose and 0.35% gellan gum (pH 5.8). Final

Acknowledgements

The 400 MHz NMR spectra were measured at the Analytical Center of Osaka University.

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