Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.

Fifteen type I terpene synthase homologs from diverse actinobacteria that were selected based on a phylogenetic analysis of more than 4000 amino acid sequences were investigated for their products. For four enzymes with functions not previously reported from bacterial terpene synthases the products were isolated and their structures were elucidated by NMR spectroscopy, resulting in the discovery of the first terpene synthases for (+)-δ-cadinol and (+)-α-cadinene, besides the first two bacterial (−)-amorpha-4,11-diene synthases. For other terpene synthases with functions reported from bacteria before the products were identified by GC–MS. The characterised enzymes include a new epi -isozizaene synthase with monoterpene synthase side activity, a 7- epi -α-eudesmol synthase that also produces hedycaryol and germacrene A, and four more sesquiterpene synthases that produce mixtures of hedycaryol and germacrene A. Three phylogenetically related enzymes were in one case not expressed and in two cases inactive, suggesting pseudogenisation in the respective branch of the phylogenetic tree. Furthermore, a diterpene synthase for allokutznerene and a sesterterpene synthase for sesterviolene were identified.

Two aspects of the biosynthesis of the non-canonical terpene synthase for 2-methylisoborneol have been studied. Several 2-methylisoborneol synthases have a proline-rich N-terminal domain of unknown function. The results presented here demonstrate that this domain leads to a reduced enzyme activity, in addition to its ability to increase long-term solubility of the protein. Furthermore, the substrate scope of the 2-methylisoborneol synthase was investigated through enzyme incubations with several substrate analogs, giving access to two C12 monoterpenoids. Implications on the stereochemical course of the terpene cyclisation by 2-methylisoborneol synthase are discussed.

The terpenoid substrate analogs (7R)-6,7-dihydrogeranylgeranyl diphosphate (6,7-dihydro-GGPP) and (7R)-6,7-dihydrogeranylfarnesyl diphosphate (6,7-dihydro-GFPP) were synthesised from (S)-citronellol and enzymatically converted with nine diterpene and two sesterterpene synthases, respectively. In two cases the substrate analogs were converted into diterpenes in cyclisation reactions corresponding to those observed for the native substrate GGPP, while the cyclisation cascade was disrupted or redirected in the other nine cases, leading to products that were named ruptenes. Several of the isolated ruptenes represent deprotonation products of cationic intermediates that are analogs of the intermediates proposed along the cyclisation cascades for the native substrates GGPP or GFPP, thus giving insights into the complex reaction mechanisms of terpene synthase mediated biosynthesis.

2-Methylisoborneol is a widespread musty odourant that is produced by many bacteria including actinomycetes, cyanobacteria and myxobacteria. Two 2-methylisoborneol synthases (MIBS) that are phylogenetically distant to the known enzyme from Streptomyces coelicolor were found to be highly active for 2-methylisoborneol biosynthesis. Based on the enzyme structure and on an amino acid sequence alignment, the MIBS from S. coelicolor was extensively studied through site-directed mutagenesis.

Mining of a terpene synthase from Streptomyces subrutilus resulted in the identification of the hexacyclic sesterterpene subrutilane, besides eight pentacyclic side products. Subrutilane represents the first case of a saturated sesterterpene hydrocarbon. Its structure, including the absolute configuration, was unambiguously determined through X-ray crystallographic analysis and stereoselective deuteration. The cyclisation mechanism to subrutilane and its side products was investigated in all detail by isotopic labelling experiments and DFT calculations. The subrutilane synthase (SrS) also converted (2Z)-GFPP into one major product. Additional compounds were obtained from the substrate analogues (7R)-6,7-dihydro-GFPP and (2Z,7R)-6,7-dihydro-GFPP with blocked reactivity at the C6-C7 bond. Interestingly, the early steps of the cyclisation cascade with (2Z)-GFPP and the saturated substrate analogues were analogous to those of GFPP, but then deviations from the natural cyclisation mode occur.

Two new N-methylated cyclopeptides, asperflomide (1) and asperflosamide (2), were obtained from the marine sponge-derived fungus Aspergillus flocculosus 16D-1. The planer structure were determined by NMR spectroscopic data and the absolute configurations of the amino acids were elucidated by the Marfey's analysis. These cyclopeptides showed weak tankyrase1/2 inhibitory activity at the centration of 40 mu M. (C) 2021 Published by Elsevier Ltd.

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.

The natural substance class of terpenoids covers an extremely wide range of different structures, although their building block repertoire is limited to the C-5 compounds DMAPP and IPP. This study aims at the characterization of methyltransferases (MTases) that modify these terpene precursors and the demonstration of their suitability for biotechnological purposes. All seven enzymes tested accepted IPP as substrate and altogether five C-6 compounds and six C-7 compounds were formed within the reactions. A high selectivity for the deprotonation site as well as high stereoselectivity could be observed for most of the biocatalysts. Only the enzyme from Micromonospora humi also accepted DMAPP as substrate, converting it into (2R)-2-methyl-IPP in vitro. In vivo studies demonstrated the production of a C-8 compound and a hydride shift step within the MTase-catalyzed reaction. Our study presents IPP / DMAPP MTases with very different catalytic properties, which provide biosynthetic access to many novel terpene-derived structures.

The biosynthesis of 2-methylisoborneol was reconstituted by elongation of dimethylallyl diphosphate (DMAPP) with (S)- and (R)-2-methylisopentenyl diphosphate (2-Me-IPP) using farnesyl diphosphate synthase (FPPS), followed by terpene cyclisation. The stereochemical course of the FPPS reaction was studied in detail using stereoselectively deuterated 2-Me-IPP isotopomers.

Both enantiomers of 2-methyllinalyl diphosphate (2-Me-LPP) were synthesized enantioselectively using Sharpless epoxidation as a key step and purification of enantiomerically enriched intermediates through HPLC separation on a chiral stationary phase. Their enzymatic conversion with 2-methylisoborneol synthase (2MIBS) demonstrates that (R)-2-Me-LPP is the on-pathway intermediate, while a minor formation of 2-methylisoborneol from (S)-2-Me-LPP may be explained by isomerization to 2-Me-GPP and then to (R)-2-Me-LPP.

Chemical investigation on a marine sponge, Dactylospongia elegans, yielded five new γ-oxygenated butenolide sesterterpene derivatives, dactylospenes A–E (1–5), as well as two known biosynthetically related compounds, luffariellolide (6) and furospinosulin B (7). The structures of these compounds were elucidated on the basis of their spectroscopic data, experimental and calculated electronic circular dichroism (ECD) analysis, as well as comparison of the NMR data with those of known analogs. These metabolites are the first γ-oxygenated butenolide sesterterpenes to be reported from this genus. These compounds were evaluated in antimicrobial, anti-inflammatory, and cytotoxic assays. Only compounds 1, 3, and 6 exhibited moderate cytotoxicity against DU145, SW1990, Huh7, and PANC-1 cancer cell lines with IC50 values in the range of 2.11–13.35 μM. Furthermore, compound 2, without cytotoxicity, exhibited significant inhibitory effects (inhibitory rate 77.5%) on nitric oxide production induced by lipopolysaccharide at 10 μM.

Four new homoverrucosane-type diterpenes (1-4) and two known analogs (5-6) were isolated from the marine sponge Halichondria sp. Their structures including absolute configurations were determined by combination of spectroscopic analyses, theoretical calculations and comparison with data of literatures. The cytotoxicity against human multiple myeloma cell line RPMI-8266 of compounds 1-4 was evaluated, but none showed activity (IC50 > 10 mu M). It was the first report that homoverrucosanes isolated from the marine sponge Halichondria sp. (C) 2020 Published by Elsevier Ltd.

Asperfloketals A (1) and B (2), two 1(10 → 6)-abeo-14,15-secosteroids featuring a novel trioxahexaheterocyclic ring system, were isolated from the sponge-associated fungus Aspergillus flocculosus 16D-1. Their structures were elucidated by extensive spectroscopic analysis and NMR chemical shifts calculations, supported by DP4+ probability analysis, and their absolute configurations were determined by ECD calculations and the modified Mosher's method. Asperfloketals A and B showed strong anti-inflammatory activity in the CuSO4-induced transgenic fluorescent zebrafish but displayed no cytotoxicity against HeLa, HepG2, and SW480 cell lines.

A novel ochratoxin-ergosteroid heterodimer, ochrasperfloroid (1), together with a known mycotoxin, ochratoxin A (2), were isolated from the sponge-derived fungus Aspergillus flocculosus 16D-1. The structure of 1 was determined on the basis of 1D/2D NMR, HRESIMS/MS, and LC-UV/MS analysis of its alkaline hydrolyzates, quantum-chemical 13C NMR calculation, and comparison with literature data. Of note, the ergosteroid embedded in 1 is also a new structure. Ochrasperfloroid (1) showed potent inhibitory activity towards IL-6 production in lipopolysaccharide (LPS)-induced THP-1 cell line, with an IC50 value of 2.02 μM, and NO production in LPS-activated RAW264.7 macrophages, with an IC50 value of 1.11 μM.

Abstract Three new sesquiterpene quinones/hydroquinones, 20-demethoxy-20-isopentylaminodactyloquinone D (1), 20-demethoxy-20-isobutylaminodactyloquinone D (2), and 19-methoxy-dictyoceratin-A (3), and five known related compounds (4−8) were isolated from the marine sponge Dactylospongia elegans. Their structures were elucidated by spectroscopic analysis, ECD calculation, single-crystal X-ray diffraction, and comparison with the literature. Compounds 3 and 5−8 exhibited activities against the human cancer cell lines DU145, SW1990, Huh7, and PANC-1 with IC50 values ranging from 2.33 to 37.85 μM. GRAPHICAL ABSTRACT

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Figure S1. UV spectrum of compound 1 Figure S2. HRESIMS spectrum of compound 1 Figure S3 HR-ESI-MS/MS spectrum of compound 1 Figure S4. 1H NMR spectrum (600 MHz, DMSO-d6) of compound 1 Figure S5. 13C NMR spectrum (150 MHz, DMSO-d6) of compound 1 Figure S6. DEPT135 Spectrum (150 MHz, DMSO-d6) of compound 1 Figure S7. HSQC spectrum of compound 1 Figure S8. HMBC spectrum of compound 1 Figure S9. 1H-1H COSY spectrum of compound 1 Figure S10 ROESY spectrum of compound 1 Figure S11 UPLC-MS analysis of D/L-FDLA derivatives of acid hydrolysate of compound 1 Figure S12 HRESIMS spectrum of compound 2 Figure S13 1H NMR spectrum (600 MHz, DMSO-d6) of compound 2 Figure S14 13C NMR spectrum (150 MHz, DMSO-d6) of compound 2 Figure S15 DEPT 135 Spectrum (150 MHz, DMSO-d6) of compound 2 Figure S16 HSQC spectrum of compound 2 Figure S17 HMBC spectrum of compound 2 Figure S18 1H-1H COSY spectrum of compound 2 Figure S19 ROESY spectrum of compound 2 Figure S20 Conformations of low-energy conformers (≥1%) of compound 1 calculated at B3LYP/6-31G(d) level of theory in MeOH (PCM). Table S1. 1H NMR (600 MHz) and 13C NMR (150 MHz) data in DMSO-d6 for 2 Table S2. The experimental and computed 13C chemical shifts for compound 1 Table S3. Comparison of experimental and computed optical rotation for compound 1 Table S4. Gibbs free energies and equilibrium populations of low-energy conformers of compound 1. Table S5. Cartesian coordinates for the low-energy reoptimized MMFF conformers (≥1%) of compound 1 calculated at B3LYP/6-31G(d) level with PCM solvent model for CH3OH. Table S6. Cartesian coordinates, Gibbs free energies (B3LYP/6-31G(d), kcal/mol) of low-energy conformers of 1 in DMSO. Table S7. Cytotoxic activity of compounds 1 and 2 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.