Biosynthesis of chlorflavonin in Aspergillus candidus. 13C- and 14C-labelling evidence for a new route to the flavonoid structure

Abstract

Aspergillus candidus growing in a glucose–leucine–salts medium synthesized chlorflavonin (1) from isotopically labelled phenylalanine, cinnamate, benzoate, or acetate. Radioactivity from [β-¹⁴C]cinnamic acid and [α-¹⁴C]-benzoic acid was incorporated less efficiently than that from L-[β-¹⁴C]phenylalanine but decarboxylation of 3-chlorosalicylic acid (3) formed by alkaline degradation of the chlorflavonin samples located all of the radioactivity at C-2 in the flavonoid. 4,5-Dimethoxyresorcinol (2), obtained by alkaline degradation of chlorflavonin labelled from sodium [1-¹⁴C]acetate and [α-¹⁴C]cinnamic acid, accounted for only part of the radioactivity. The label from both precursors was distributed between ring A and one or more of the C-3, C-4, and C-3 methoxy-carbon atoms. ¹³C N.m.r. spectrometry of chlorflavonin labelled from sodium [1-¹³C]acetate showed ¹³C incorporation into C-4, C-5, C-7, and C-8a. Sodium [1-¹³C_0;1;1,2-¹³C_1;0;1]acetate was incorporated intact into (C-3,C-4) and all adjacent pairs of ring-A carbon atoms. The results indicate a pathway of flavonoid biosynthesis differing from that of higher plants in that a C₆-C₁ precursor unit is condensed with four C₂ units. In the route proposed, the heterocyclic ring is formed before ring A is substituted at C-8 and while it is free to rotate at the enzyme surface.