Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products

Pericyclic reactions are powerful transformations for the construction of carbon–carbon and carbon–heteroatom bonds in organic synthesis. Their role in biosynthesis is increasingly apparent, and mechanisms by which pericyclases can catalyse reactions are of major interest 1 . [4+2] cycloadditions (Diels–Alder reactions) have been widely used in organic synthesis 2 for the formation of six-membered rings and are now well-established in biosynthesis 3 – 6 . [6+4] and other ‘higher-order’ cycloadditions were predicted 7 in 1965, and are now increasingly common in the laboratory despite challenges arising from the generation of a highly strained ten-membered ring system 8 , 9 . However, although enzyme-catalysed [6+4] cycloadditions have been proposed 10 – 12 , they have not been proven to occur. Here we demonstrate a group of enzymes that catalyse a pericyclic [6+4] cycloaddition, which is a crucial step in the biosynthesis of streptoseomycin-type natural products. This type of pericyclase catalyses [6+4] and [4+2] cycloadditions through a single ambimodal transition state, which is consistent with previous proposals 11 , 12 . The [6+4] product is transformed to a less stable [4+2] adduct via a facile Cope rearrangement, and the [4+2] adduct is converted into the natural product enzymatically. Crystal structures of three pericyclases, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis establish the mechanism of this transformation. This work shows how enzymes are able to catalyse concerted pericyclic reactions involving ambimodal transition states. Enzymatic catalysis of pericyclic [6+4] cycloaddition reactions to form ten-membered rings is observed during biosynthesis of the macrocyclic antibiotic streptoseomycin, and the mechanism of these transformations is established.