A Branched Diterpene Cascade: The Mechanism of Spinodiene Synthase from Saccharopolyspora spinosa

A diterpene synthase from Saccharopolyspora spinosa was found to convert geranylgeranyl diphosphate into the new natural products spinodiene A and B, accompanied by 2,7,18‐dolabellatriene. The structures and the formation mechanism of the enzyme products were investigated by extensive isotopic labelling experiments, which revealed an unusual branched isomerisation mechanism towards the neutral intermediate 2,7,18‐dolabellatriene. A Diels–Alder reaction was used to convert the main diterpene product with its rare conjugated diene moiety into formal sesterterpene alcohols.     

Spiroluchuene A Synthase: A Cyclase from Aspergillus luchuensis Forming a Spirotetracyclic Diterpene

The diterpene synthase AlTS was identified from Aspergillus luchuensis. AlTS catalyses the formation of the diterpene hydrocarbon spiroluchuene A, which exhibits a novel skeleton characterised by a spirocyclic ring system. The cyclisation mechanism towards this compound was elucidated through isotopic labelling experiments in conjunction with DFT calculations and metadynamic simulations. The biosynthetic intermediate luchudiene, besides the derivative spiroluchuene B, was captured from an enzyme variant obtained through site-directed mutagenesis. With its 10-membered ring luchudiene is structurally related to germacrenes and can undergo a Cope rearrangement to luchuelemene.     

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

Di‐ and sesterterpene synthases produce C₂₀ and C₂₅ isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene ( 1 ), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.     

Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane-Type Diterpenoids

CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane-type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for the unique carbocation-driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane-type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.     

Discovery and Characterization of a New Family of Diterpene Cyclases in Bacteria and Fungi

Diterpene cyclases from bacteria and basidiomycete fungi are seldom studied. Here, we presented the identification and verification of EriG, a member of the UbiA superfamily, as the enzyme responsible for the cyclization of the cyathane skeleton in the mushroom Hericium erinaceum . Genome mining using the EriG protein sequence as a probe led to the discovery of a new family of ubiquitous UbiA‐related diterpene cyclases in bacteria and fungi. We successfully characterized seven new diterpene cyclases from bacteria or basidiomycete fungi with the help of an engineered Escherichia coli strain and determined the structures of their corresponding products. A new diterpene with an unusual skeleton was generated during this process. The discovery of this new family of diterpene cyclases provides new insight into the UbiA superfamily.     

Diterpene Biosynthesis in Actinomycetes: Studies on Cattleyene Synthase and Phomopsene Synthase

Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.     

Two Bacterial Diterpene Synthases from Allokutzneria albata Produce Bonnadiene,Phomopsene, and Allokutznerene

Two diterpene synthases from Allokutzneria albata were studied for their products, resulting in the identification of the new compound bonnadiene from the first enzyme. Although phylogenetically unrelated to fungal phomopsene synthase, the second enzyme produced a mixture of phomopsene and a biosynthetically linked new compound, allokutznerene, as well as spiroviolene. Both enzymes were subjected to in‐depth mechanistic studies involving isotopic labelling experiments, metal‐cofactor variation, and site‐directed mutagenesis. Oxidation products of phomopsene and allokutznerene are also discussed.     

New adduct of abietane-type diterpene from Salvia leriifolia Benth.

A new adduct of abietane-type diterpene, salvialeriicone (1), was isolated from Salvia leriifolia Benth., along with a new chemical entity nor-abietane diterpene, 2-isopropyl-8,8-dimethyl-7,8-dihydrophenanthrene-1,4,5(6H)-trione (2). Their structures were determined using mass spectrometry, and 1D- and 2D-NMR spectroscopy.     

Two New Acidic Diterpenoids from the Heartwood of Pinus massoniana Lamb.

A new abietane diterpene, named abietopinoic acid, and a new podocarpane diterpene, named podopinoic acid, were isolated from the acetone extract of the heartwood of Pinus massoniana. Their structures were established as 12‐hydroxy‐7‐oxoabieta‐8,11,13‐trien‐18‐oic acid ( 1 ) and 13‐hydroxy‐7‐oxopodocarpane ( 2 ) by means of spectroscopic analyses including 2D‐NMR. To the best of our knowledge, this is the second report of a podocarpa‐8,11,13‐trien‐18‐oic acid diterpene, isolated from the genus Pinus.     

Clerodane and ent‐Kaurane Diterpene Glycosyl and Glycoside Derivatives from the Leaves of Casearia sylvestris

Five new clerodane diterpene glycosyl derivatives, caseariasides A–E ( 1 – 5 , resp.) and three new ent‐kaurane diterpene glucosides, sylvestrisides C–E ( 6 – 8 , resp.) were isolated from the leaves of Casearia sylvestris. Their structures were determined on the basis of chemical and spectroscopic analyses.     

Mechanistic Characterisation and Engineering of Sesterviolene Synthase from Streptomyces violens

The sesterviolene synthase from Streptomyces violens was identified and represents the second known sesterterpene synthase from bacteria. Isotopic labelling experiments in conjunction with DFT calculations were performed that provided detailed insight into its complex cyclisation mechanism. Enzyme engineering through site-directed mutagenesis gave access to a high-yielding enzyme variant that provided six additional minor products and the main product in sufficient quantities to study its chemistry.     

Labdane Diterpenes and Flavonoids from Leonurus japonicus

Chemical investigation of the aerial part of Leonurus japonicus led to the isolation and characterization of a new labdane‐type diterpene, named 3α‐acetoxy‐15‐O‐methylleopersin C ( 3 ), and a new acylated rutin derivative, named 2′′′‐syringylrutin ( 9 ), along with seven known compounds, including a labdane‐type diterpene and six flavonoids. The structures of the new compounds were established by spectroscopic methods.     

A new tigliane-type diterpene from Euphorbia dracunculoides Lam

One new tigliane-type diterpene, 4-deoxy-4(β)H-8-hydroperoxyphorbol-12-benzoate-13-isobutyrate (1), together with two known diterpenoids, 3-acetyl-5,8-dibenzoyl-14α-propanoyl-13,17-epoxy-7-myrsinaone diterpene with C9–C10 cyclised to form an additional lactone ring (2), Euphodendriane A (3) have been isolated from the whole plants of Euphorbia dracunculoides Lam. Their structures were elucidated by means of extensive spectroscopic analysis (NMR and HR-ESI-MS) and comparison with data reported in the literature. This is the first isolation of 8-hydroperoxy tigliane diterpene (1) from the genus of Euphorbia. All compounds were evaluated for their antifungal activities.     

Two New, 1‐Oxygenated ent‐Kaurane‐Type Diterpenes from Croton kongensis

One new ent‐8,9‐secokaurane diterpene, kongensin D ( 1 ), and one new ent‐kaurane diterpene, kongensin E ( 2 ), along with one known compound, (7α)‐7,18‐dihydroxy‐ent‐kaur‐16‐en‐15‐one 18‐acetate ( 3 ), were isolated from the aerial parts of Croton kongensis. The structures of the new compounds were elucidated by means of HR‐MS and in‐depth NMR experiments, and by comparison with literature data. Compounds 1 and 2 showed an unusual oxygenation pattern with an OH or AcO group at C(1).     

A New ent‐Kaurane Diterpene from Stems of Alibertia macrophylla K. Schum. (Rubiaceae)

Phytochemical investigations of the stems of a specimen of Alibertia macrophylla led to the isolation and characterization of the new diterpene ent‐kaurane‐2β,3α,16α‐triol ( 1 ), along with triterpenes 2 – 8 , iridoids 9 – 12 , and phenolic acids 13 – 15 . The structure of 1 was established based on spectroscopic studies (¹H‐ and ¹³C‐NMR, IR, and HR‐ESI‐MS). This is the first report of the isolation of a diterpene from the Alibertia genus in Rubiaceae.     

A New Glycoside and a Novel‐Type Diterpene from Hemerocallis fulva (L.) L.

The CHCl₃ extract of dried roots of Hemerocallis fulva (L.) L. afforded a novel diterpene named hemerocallal A ( 1 ), which is the second reported naturally occurring diterpene with a trans‐bicyclo[5.1.0]octane system. The BuOH extract afforded a new glycoside named hemerocalloside ( 2 ). Their structures were established on the basis of spectroscopic and chemical studies.     

New Diterpene Alkaloids from Aconitum sungpanense var. leucanthum

Five new C₁₉ diterpene alkaloids, leucanthumsines A ( 1 ), B ( 2 ), C ( 3 ), D ( 4 ), and E ( 5 ), were isolated from the Chinese medicinal herb Aconitum sungpanense var. leucanthum, together with the known C₁₉ diterpene alkaloids pseudaconine, neoline, 1‐O‐methyldelphisine, crassicaudine, chasmanine, talatisamine, indaconitine, ezochansmanine, and leueantine D. The structures of these new alkaloids were elucidated by HR‐MS and advanced NMR methods, including ¹H‐ and ¹³C‐NMR (DEPT), ¹H,¹H‐COSY, HMQC, and HMBC experiments.     

A New Alkaloid from the Roots of Aconitum carmichaeli Debx.

A new diterpene alkaloid, 12‐epi‐15‐O‐acetyl‐17‐benzoyl‐16‐hydroxy‐16,17‐dihydronapelline ( 1 ), along with nine known diterpene alkaloids including yunaconitine ( 2 ), neoline ( 3 ), mesaconitine ( 4 ), beiwutine ( 5 ), chasmanine ( 6 ), songorine ( 7 ), 12‐epi‐napelline ( 8 ), foresticine ( 9 ), and 15α‐hydroxyneoline ( 10 ) were isolated from the roots of Aconitum carmichaeli Debx. The structure of compound 1 was elucidated by comprehensive spectroscopic analysis.     

Two new diterpene and sesquiterpene from Pteris dispar

A new diterpene and a new sesquiterpene were isolated from Pteris dispar and identified on the basis of spectral data including 2D NMR.Compounds 1 and 2 showed potent cytotoxic activity against KB cell.     

Using Terpene Synthase Plasticity in Catalysis: On the Enzymatic Conversion of Synthetic Farnesyl Diphosphate Analogues

Four synthetic farnesyl diphosphate analogues were enzymatically converted with three bacterial sesquiterpene synthases, including β-himachalene synthase (HcS) and (Z)-γ-bisabolene synthase (BbS) from Cryptosporangium arvum, and germacrene A synthase (SmTS6) from Streptomyces mobaraensis. These enzyme reactions not only yielded several previously unknown compounds, showing that this approach opened the door to a new chemical space, but substrates with blocked or altered reactivities also gave interesting insights into the cyclisation mechanisms and the potential to catalyse reactions with different initial cyclisation modes.