Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1.

Elatenyne is a marine natural product that was isolated in 1986. Despite its simple 2,2'-bifuranyl backbone, its relative structure was only recently determined. The absolute configuration of elatenyne has still not been unequivocally confirmed because of its pseudo-meso core structure, which results in a specific rotation, [α]D  , of almost zero. In this work, the structure of natural elatenyne was determined by the crystalline sponge method and the use of a porous coordination network (a crystalline sponge) capable of absorbing organic guests; in the sponge, the absorbed guests are ordered and crystallographically observable. The crystalline sponge could differentiate between the two very similar alkyl side chains, and the absolute structure of elatenyne was thus reliably determined. The total amount required for the experiments was only approximately 100 μg, and the majority (95 μg) could be recovered after the experiments.

Sesquiterpene scaffolds are the core backbones of many medicinally and industrially important natural products. A plethora of sesquiterpene synthases, widely present in bacteria, fungi, and plants, catalyze the formation of these intricate structures often with multiple stereocenters starting from linear farnesyl diphosphate substrates. Recent advances in next-generation sequencing and metabolomics technologies have greatly facilitated gene discovery for sesquiterpene synthases. However, a major bottleneck limits biochemical characterization of recombinant sesquiterpene synthases: the absolute structural elucidation of the derived sesquiterpene products. Here, we report the identification and biochemical characterization of LphTPS-A, a sesquiterpene synthase from the red macroalga Laurencia pacifica. Using the combination of transcriptomics, sesquiterpene synthase expression in yeast, and microgram-scale nuclear magnetic resonance-coupled crystalline sponge X-ray diffraction analysis, we resolved the absolute stereochemistry of prespatane, the major sesquiterpene product of LphTPS-A, and thereby functionally define LphTPS-A as the first bourbonane-producing sesquiterpene synthase and the first biochemically characterized sesquiterpene synthase from red algae. Our study showcases a workflow integrating multiomics approaches, synthetic biology, and the crystalline sponge method, which is generally applicable for uncovering new terpene chemistry and biochemistry from source-limited living organisms.

Characterization of complex natural product mixtures to the absolute structural level of their components often requires significant amounts of starting materials and lengthy purification process, followed by arduous structure elucidation efforts. The crystalline sponge (CS) method has demonstrated utility in the absolute structure elucidation of isolated organic compounds at miniscule quantities compared to conventional methods. In this work, we developed a new CS-based workflow that greatly expedites the in-depth structural analysis of crude natural product extracts. Using a crude extract of the red alga Laurencia pacifica, we showed that CS affinity screening prior to compound isolation enables prioritization of analytes present in the extract, and we subsequently resolved the molecular structures of six sesquiterpenes with stereochemical clarity from around 10 mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples.

An enantio-, diastereo-, regio-, and chemoselective phosphine-catalyzed β,γ-umpolung domino reaction of allenic esters with dienones has been developed for the first time. The designed sequence, involving oxy-Michael and Rauhut-Currier reactions, produced highly functionalized tetrahydrobenzofuranones, bearing a chiral tetrasubstituted stereogenic center, in up to 96 % ee.