Enzymatic [4+2] Cycloadditions in the Biosynthesis of Spirotetramates and Spirotetronates

The Diels-Alder reaction is a quintessential type of [4+2] cycloaddition that remains one of the most intriguing transformations in synthetic chemistry. This reaction has long been envisaged to participate in the biosynthesis of a number of cyclohexene-containing natural products, although the question of whether a bona fide Diels-Alderase exists remains unsolved. In nature, there are remarkably few enzymes known to have the activity of [4+2] cycloaddition. These enzymes are phylogenetically distinct and are often classified according to the specific chemical structures. The variation of protein ancestors and in many cases the instability/complexity of the substrates and products pose a significant challenge in identification of the [4+2] cycloaddition catalysts using general homology-based mining approaches. We here provide the detailed description of the multiple comparison-based strategy and methods for the characterization of two distinct types of dedicated [4+2] cyclases (eg, PyrE3 and PyrI4) in the biosynthesis of spirotetramates and spirotetronates, where they act in tandem for coordinated cross-bridging of a linear polyene intermediate into a enantiomerically pure pentacyclic core. The search of new protein scaffolds with the [4+2] cycloaddition activity could enrich the pool of the candidates for mechanistic examination of a true enzymatic Diels-Alder reaction. The protocols presented in this study would also be applicable to the study of other functionally similar but phylogenetically different proteins, eg, the spiroketal cyclases.