Polyketide Cross-Bridging and Natural Product Biosynthesis
The Diels-Alder reaction is an efficient cyclization reaction between a conjugated diene and a dienophile to form a substituted cyclohexene ring with high regio- and stereoselectivity, and has been employed extensively in organic synthesis. This [4 + 2] cycloaddition reaction has also been proposed to be a key transformation in the biosynthesis of many natural products. Several enzymes whose functions may be considered as “Diels-Alderases,” such as lovastatin nonaketide synthase, solanapyrone synthase, and macrophomate synthase, have been identified and studied. However, doubts about whether their mechanisms are true Diels-Alder type reaction remain. In an attempt to find and present more evidence for the existence of “Diels-Alderases” in Nature, we are currently investigating the biosynthesis of two bacterial metabolites, spinosyn A (1) and kijanimicin (2), in which the cross-bridging reaction in each case may be a Diels-Alder reaction.
Spinosyns are macrolide-type natural products isolated from Saccaropolyspora spinosa. The basic structural components of spinosyns consist of a tetracyclic macrolactone (3) and two deoxy sugars, D-forosamine (4) and methylated L-rhamnose (5). A mixture of spinosyn A and D, bearing a trade name Tracer™, a commercial product of Dow AgroSciences, is used as an insect control agent. Spinosyns have been shown to be benign to non-target organisms. Analysis of the spinosyn biosynthetic gene cluster allows the deduction of the structure of the initial macrolactone precursor (6). The proposed cross-bridging reaction (6 → 7) to generate the tetracyclic ring skeleton (7) can be envisioned to involve a Diels-Alder cyclization.
Kijanimicin (2) is a spirotetronate class polyketide antibiotic isolated from Actinomadura kijaniata. It is effective against some gram-positives bacteria, anaerobes, and malaria. It also possesses antitumor activity. Kijanimicin is composed of a pentacyclic core containing an unusual spirotetronic acid moiety, which is decorated by five deoxysugars, including four digitoxose (8) and the unusual nitro sugar D-kijanose (9). Analysis of the possible biosynthetic pathway for kijanimicin indicates the potential involvement of two Diels-Alder cyclization reactions in the construction of the pentacyclic aglycone.
Using a combination of chemical, biochemical, spectroscopic, and genetic methods, we have cloned and sequenced the kijanimicin biosynthetic gene cluster and are now working on biochemical characterization of enzymes involved in polyketide cross-bridging reactions. In particular, we would like to gain evidence for enzyme-catalyzed Diels-Alder reactions. More generally, we are interested in the biosynthesis of bioactive compounds possessing novel structural features for which the biosynthetic pathways and mechanisms of enzymatic transformations are not well understood. A multi-faceted approach is employed to study these problems.