Astellifadiene: Structure Determination by NMR Spectroscopy and Crystalline Sponge Method, and Elucidation of its Biosynthesis

Abstract

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.

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution

“…4). Feeding experiments with 13 C-labeled precursors in fungi have suggested the formation of a quiannulatene scaffold via a 5/12/5 tricyclic carbocation and an intriguing cyclobutane intermediate (15,42). With the isolation of tricyclic (5/12/5) and tetracyclic (5/8/6/5) carbocation-derived (−)-variculatriene A (5) and (−)-aleurodiscalene A (9) as minor products of AtTPS25 and Bo250, which produce mainly the quiannulatene scaffold sesterterpenes 4 and 8, respectively (Fig.…”

“…4. Although the astellatene and retigeranin B scaffolds have recently been proposed to also be derived from 5/12/5 tricyclic carbocations (12), previous feeding experiments with 13 C-labeled precursor (43) and our finding that the 11/6/5 scaffold (2) is the major product of AtTPS18 and is present as a very minor product of AtTPS19, -25, and -30 ( Fig. 2C) suggests that the bicyclic C12 carbocation may be diverged into tricyclic 5/12/5 and 11/6/5 cations en route to the formation of pentacyclic sesterterpenes, such as (−)-retigeranin B (3) and (+)-astellatene (6).…”

“…Enone 11 was synthesized by allylic oxidation with pyridinium dichromate (PDC) and tert-butyl hydroperoxide (TBHP) in dichloromethane (DCM). Crystal structures (10)(11)(12)(13) are presented with displacement ellipsoids shown at 50% probability.…”

“…In fungi, six STSs have recently been identified and shown to synthesize ophiobolin F (in Aspergillus clavatus) (11), sesterfisherol (in Neosartorya fischeri) (12), and stellata-2,6,19-triene, (2E)-α-cericerene, quiannulatene, and astellifadiene (in Emericella variecolor) (13)(14)(15)(16) (Fig. 1A).…”

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes

“…Furthermore, this has been extended to two more clade B-bifunctional terpene synthases, that is, astellifadiene synthase and stellatatriene synthase, which have recently been identified. 19,20 In this study, we developed an efficient method to synthesize sitespecifically isotope-labeled oligoprenyl diphosphate precursors, such as DMAPP, GPP, FPP and GGPP, starting from four chemically synthesized [ 2 H]-IPPs by enzymatic transformation. By using these precursors, we demonstrated that the MS-based analysis of sitespecifically isotope-labeled terpenes provides sufficient information to elucidate the cyclization mechanism catalyzed by PaPS.…”