2D NMR-spectroscopic screening reveals polyketides in ladybugs

Abstract

Small molecules of biological origin continue to yield the most promising leads for drug design, but systematic approaches for exploring nature's cache of structural diversity are lacking. Here, we demonstrate the use of 2D NMR spectroscopy to screen a library of biorationally selected insect metabolite samples for partial structures indicating the presence of new chemical entities. This NMR-spectroscopic survey enabled detection of novel compounds in complex metabolite mixtures without prior fractionation or isolation. Our screen led to discovery and subsequent isolation of two families of tricyclic pyrones in Delphastus catalinae, a tiny ladybird beetle that is employed commercially as a biological pest control agent. The D. catalinae pyrones are based on 23-carbon polyketide chains forming 1,11-dioxo-2,6,10-trioxaanthracene and 4,8-dioxo-1,9,13-trioxaanthracene derivatives, representing ring systems not previously found in nature. This study highlights the utility of 2D NMR-spectroscopic screening for exploring nature's structure space and suggests that insect metabolomes remain vastly underexplored.

Fig. 1.

Delphastus catalinae. (A) Adult beetle next to rim of a US one cent coin and catalipyrone A (1). (B) Pupa coated with glandular hairs (arrow) emerging from larval skin.

Fig. 2.

Overview of the use of NMR-spectroscopic screening in the discovery of the catalipyrones. Ten largely unfractionated insect metabolite extracts were characterized using 2D NMR spectroscopy, principally high-resolution dqfCOSY spectra. Analysis of the dqfCOSY spectra produced libraries of partial structures. To assess the likelihood of finding NCEs, these partial structures and their NMR-spectroscopic data were compared against available databases. For additional detail on the proposed partial structures, see SI Appendix, Figs. S1–S4 and Table S1.

Fig. 3.

Comparison of dqfCOSY spectra obtained for pupal and adult D. catalinae metabolite extracts, illustrating the utility of high-resolution 2D NMR spectra for the comparative analysis of complex metabolite mixtures. (A) 0.8- to 3.4-ppm section of the spectrum obtained for D. catalinae pupae (600 MHz, CD₂Cl₂), illustrating the level of assignment achieved for this sample. Signals were assigned to different compound classes based on detailed analysis of this dqfCOSY plus HMBC and HSQC spectra. Cross-peaks that could be assigned are boxed; solid pink: germacrene-type sesquiterpenoids; dashed pink: humulene-type sesquiterpenoids; dashed black and blue: farnesol; dashed green: fatty acids. Polyketides: blue (8), orange (6 and isomer), green (10), and black (shared signals of 8–10). (B) Corresponding section of spectrum obtained for D. catalinae adults. The spectrum reveals a large number of polyketide fragments different from those observed in the pupae (1–7, boxed red), in addition to terpenoids and lipids similar to those found in A. See SI Appendix for additional detail.

Fig. 4.

Structures of catalipyrones A–J (1–10) from D. catalinae. (A) The 1,11-dioxo-2,6,10-trioxa-anthracenes 1–7 were identified from adult beetles, whereas the 4,8-dioxo-1,9,13-trioxaanthracenes 8–10 are present in the pupal defensive secretion. The stereochemistry of the side chains in 2–9 is proposed in analogy to that determined for 1 (see text). (B) Model for the biosynthesis of 1,11-dioxo-2,6,10-trioxa-anthracenes and 4,8-dioxo-1,9,13-trioxaanthracenes 1–10 from two similarly functionalized tripropionate units (red) and one 5-carbon unit (blue).