Synthesis of model cytochalasan precursors and in vitro studies of their activities

Abstract

Cytochalasans are one type of PKS-NRPS secondary metabolites. The tailoring steps involved in cytochalasan biosynthesis are almost clear, but the formation of the isoindolone core is still mysterious. The ACE1 metabolite probably belongs to the cytochalasan family based on the biosynthetic gene cluster (BGC) analysis, even though the structure is not yet elucidated. Heterologous expression of ace1 (PKS-NRPS) and rap1 (trans-ER) in A. oryzae produced an unexpected alcohol, which is unlikely to undergo further Knoevenagel reaction. Therefore it is deduced to be a shunt metabolite during heterologous expression. To investigate the biosynthesis of the cytochalasan skeleton including reductive release, putative Knoevenagel condensation and Diels-Alder reaction, three model compounds mimicking the ACE1 metabolite precursor were designed, prepared and applied for in vitro assays. Model A was designed and synthesized for over-reduction investigation. In vitro assays confirmed that model A can be reduced to the corresponding primary alcohol by A. oryzae cell free extract. This indicates that the alcohol is a shunt metabolite and A. oryzae is not a suitable host for cytochalasan heterologous expression. Model B was designed and prepared to study Knoevenagel condensation. In vitro assays with model B suggested model B can possibly form pyrrolinone spontaneously in buffer, while pyrrolinone was converted rapidly to alcohols. However, no enzyme activity was observed. Besides, pyrrolinone tautomerism was further investigated by comparing tautomerized pyrrolinone and non-tautomerised pyrrolinone. From NMR and UV analysis, it is confirmed that pyrrolinones tautomerises and are no longer competitive for the following Diels-Alder reaction. Model C was designed for both Knoevenagel condensation and Diels-Alder reaction tests. In vitro Assays showed that pyrrolinone was formed spontaneously, further confirming the Knoevenagel condensation is spontaneous. But its corresponding pyrrolinone also tautomerised and is no longer suitable for the following Diels-Alder reaction.