Biosynthesis of rotenone and amorphigenin. Study of the origins of isopropenyl-substituted dihydrofuran E-rings using isotopically labelled late precursors

Abstract

Whilst epoxidation of rot-2′-enonic acid is the most likely source of dalpanol in Amorpha fruticosa seedlings, administration of (5′R,6′S)-[7′-³H]dalpanol shows that it is not an intermediate on the path to rotenone and amorphigenin. Labelled 4′-hydroxy- or 5′-hydroxy-rot-2′-enonic acid also do not qualify as intermediates in rotenone biosynthesis, but they are each converted into amorphigenin with chemospecific attack on the methyl group. By administration and re-isolation of [8′-¹⁴C]amorphigenin from A. fruticosa seedlings, our earlier conclusion that hydroxylation of rotenone to form amorphigenin proceeds with even label scrambling between C-7′ and C-8′, probably via an allylic radical, is confirmed. Competitive double-labelling experiments are employed to support a scheme in which rotenone derives directly from rot-2′-enonic acid by an enzyme-induced radical-type reaction without the intervention of an hydroxylated intermediate, and the two labelled hydroxyrot-2′-enonic acids are similarly cyclised using their methyl groups. The incorporations into amorphigenin of labelled 4- and 5-hydroxyrot-2′-enonic acids, both of which are shown to occur naturally in A. fruticosa, are similar, but only about one sixth that of rotenone.

This, and our related biosynthetic work, rests on an extensive programme of isotopic labelling and reconstructive synthesis. Our earlier method for making [7′-¹⁴C]-rotenone has been improved, and similar procedures adapted for [7′-¹³C]- and [7′-¹⁴C]-amorphigenin, 8′-Labelled rotenones are made by a positional interchange using addition of benzeneselenenyl chloride and elimination of the selenoxide, whilst [8′-¹⁴C]amorphigenin is made via addition of phenylselenophthalimida. Unlabelled amorphigenin can be isotopically labelled by oxidation to the aldehyde and reduction using sodium borodeuteride or borotritide and a method additional to those we have described earlier is given for tritium labelling of rot-2′-enonic acid. [¹³C]- and [¹⁴C]-Labelling in the 5′-position of 4′-and 5′-hydroxyrot-2′-enonic acids can be attained through the catalytic hydrogenolysis of [7′-¹⁴C]amorphigenin though special methods must be used to scrub the samples totally free from the latter. Methods based on the hydrolysis of labelled 4′-bromorot-2′-enonic acid are also described, and 4′-tritium-labelled 4′-hydroxyrot-2′-enonic acid is made from unlabelled material, or from rot-2′-enonic acid, by simple oxidation/reduction methods.