Streptomyces sungeiensis SD3 as a Microbial Chassis for the Heterologous Production of Secondary Metabolites.

We present the newly isolated Streptomyces sungeiensis SD3 strain as a promising microbial chassis for heterologous production of secondary metabolites. S. sungeiensis SD3 exhibits several advantageous traits as a microbial chassis, including genetic tractability, rapid growth, susceptibility to antibiotics, and metabolic capability supporting secondary metabolism. Genomic and transcriptomic sequencing unveiled the primary metabolic capabilities and secondary biosynthetic pathways of S. sungeiensis SD3, including a previously unknown pathway responsible for the biosynthesis of streptazone B1. The unique placement of S. sungeiensis SD3 in the phylogenetic tree designates it as a type strain, setting it apart from other frequently employed Streptomyces chassis. This distinction makes it the preferred chassis for expressing biosynthetic gene clusters (BGCs) derived from strains within the same phylogenetic or neighboring phylogenetic clade. The successful expression of secondary biosynthetic pathways from a closely related yet slow-growing strain underscores the utility of S. sungeiensis SD3 as a heterologous expression chassis. Validation of CRISPR/Cas9-assisted genetic tools for chromosomal deletion and insertion paved the way for further strain improvement and BGC refactoring through rational genome editing. The addition of S. sungeiensis SD3 to the heterologous chassis toolkit will facilitate the discovery and production of secondary metabolites.

Biosynthesis of Octacosamicin A: Uncommon Starter/extender Units and Product Releasing via Intermolecular Amidation.

Octacosamicin A is an antifungal metabolite featuring a linear polyene-polyol chain flanked by N-hydroxyguanidine and glycine moieties. We report here that sub-inhibitory concentrations of streptomycin elicited the production of octacosamicin A in Amycolatopsis azurea DSM 43854T . We identified the biosynthetic gene cluster (oca BGC) that encodes a modular polyketide synthase (PKS) system for assembling the polyene-polyol chain of octacosamicin A. Our analysis suggested that the N-hydroxyguanidine unit originates from a 4-guanidinobutyryl-CoA starter unit, while the PKS incorporates an α-hydroxyketone moiety using a (2R)-hydroxymalonyl-CoA extender unit. The modular PKS system contains a non-canonical terminal module that lacks thioesterase (TE) and acyl carrier protein (ACP) domains, indicating the biosynthesis is likely to employ an unconventional and cryptic off-loading mechanism that attaches glycine to the polyene-polyol chain via an intermolecular amidation reaction.

Forrestiacids E-K: Further [4 + 2]-Type Triterpene-Diterpene Hybrids as Potential ACL Inhibitors from the Vulnerable Conifer Pseudotsuga forrestii.

Seven [4 + 2]-type triterpene-diterpene hybrids derived from a rearranged or a normal lanostane unit (dienophile) and an abietane moiety (diene), forrestiacids E-K (1-7, respectively), were further isolated and characterized from Pseudotsuga forrestii (a vulnerable conifer endemic to China). The intriguing molecules were revealed with the guidance of an LC-MS/MS-based molecular ion networking strategy combined with conventional phytochemical procedures. Their chemical structures with absolute configurations were established by spectroscopic data, chemical transformation, electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. They all contain a rare bicyclo[2.2.2]octene motif. Both forrestiacids J (6) and K (7) represent the first examples of this unique class of [4 + 2]-type hybrids that arose from a normal lanostane-type dienophile. Some isolates remarkably inhibited ATP-citrate lyase (ACL), with IC50 values ranging from 1.8 to 11 µM. Docking studies corroborated the findings by highlighting the interactions between the bioactive compounds and the ACL enzyme (binding affinities: -9.9 to -10.7 kcal/mol). The above findings reveal the important role of protecting plant species diversity in support of chemical diversity and potential sources of new therapeutics.

Unprecedented spirodioxynaphthalenes from the endophytic fungus Phyllosticta ligustricola HDF-L-2 derived from the endangered conifer Pseudotsuga gaussenii.

Four undescribed palmarumycin-type spirodioxynaphthalenes (phyligustricins A-D) and a known biogenetic precursor (palmarumycin BG1) were isolated from a solid fermentation of Phyllosticta ligustricola HDF-L-2, an endophyte associated with the endangered Chinese conifer Pseudotsuga gaussenii. The structures were elucidated by spectroscopic methods, single-crystal X-ray diffraction analyses, and electronic circular dichroism calculations. Both phyligustricins A and B have an unprecedented spirodioxynaphthalene-derived skeleton containing an extra 4H-furo [3,2-c]pyran-4-one moiety, while phyligustricins C and D are p-hydroxy-phenethyl substituted spirodioxynaphthalenes. The plausible biosynthetic relationships of the isolates were briefly proposed. Phyligustricins C and D and palmarumycin BG1 showed considerable antibacterial activity against Staphylococcus aureus, each with an MIC value of 16 µg/mL. Palmarumycin BG1 displayed significant inhibitory effects against ACL and ACC1, with IC50 values of 1.60 and 8.00 µM, respectively.

Antibacterial and antibiofilm efficacy of the preferred fractions and compounds from Euphorbia humifusa (herba euphorbiae humifusae) against Staphylococcus aureus.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia humifusa Willd., known as Di-Jin-Cao in Chinese, has long been utilized as a traditional herb for the treatment of furuncles and carbuncles mainly caused by Staphylococcus aureus infection. Despite extensive chemical and pharmacological studies reported previously for E. humifusa, the antibacterial and antibiofilm activities against S. aureus as well as the related mechanism of action (MoA) remain largely obscure. AIM OF THE STUDY: To investigate the antibacterial and antibiofilm activities of the preferred fractions and compounds from E. humifusa against S. aureus and assess the associated MoA. MATERIALS AND METHODS: The bioactive fractions and compounds were obtained from the 75% ethanol extract of E. humifusa (75%-EEEH) with the assistance of the related antibacterial and antibiofilm screening. Their antibacterial activities were determined using the broth microdilution method, whilst the inhibition of biofilm formation and the disruption of preformed biofilm were assessed by crystal violet staining and confocal laser scanning microscopy (CLSM). To achieve more effective therapies, the combinatory effects of different components were also studied. The biofilm metabolic activities of isolated compounds were evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The scanning electron microscopy (SEM) and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to explore the antibiofilm mechanism. RESULTS: Fractions DJC06 and DJC07 collected from the ethyl acetate extract of the 75%-EEEH exhibited antibacterial activity (MIC = 256 µg/mL) against S. aureus and further separation of these two fractions led to the isolation and characterization of 22 compounds. Among the isolates, luteolin (LU), quercetin (QU), and kaempferol (KA) are the verified components associated with the antibacterial and antibiofilm activities by displaying individual or combinational MIC values of 8-128 µg/mL and 70.9-99.7% inhibition for biofilm formation. Importantly, QU and KA can work in synergy with LU to significantly enhance the efficacy via destroying cell integrity, increasing membrane permeability, and down-regulating the biofilm-related gene expression. CONCLUSIONS: The preferred fractions and compounds from E. humifusa exerted desired antibacterial and antibiofilm efficacy against S. aureus via a MoA involving cell morphology disruption and altered genes expression. The findings herein not only support its traditional use in the treatment of furuncles and carbuncles, but reveal E. humifusa is a potential source for producing promising antibiofilm alternatives against S. aureus and highlight the isolated components (LU, QU, KA) that can potentiate the efficacy when used in synergy.

Characterization of the Biosynthetic Gene Cluster and Shunt Products Yields Insights into the Biosynthesis of Balmoralmycin.

Angucyclines are a family of structurally diverse, aromatic polyketides with some members that exhibit potent bioactivity. Angucyclines have also attracted considerable attention due to the intriguing biosynthetic origins that underlie their structural complexity and diversity. Balmoralmycin (compound 1) represents a unique group of angucyclines that contain an angular benz[α]anthracene tetracyclic system, a characteristic C-glycosidic bond-linked deoxy-sugar (d-olivose), and an unsaturated fatty acid chain. In this study, we identified a Streptomyces strain that produces balmoralmycin and seven biosynthetically related coproducts (compounds 2-8). Four of the coproducts (compounds 5-8) are novel compounds that feature a highly oxygenated or fragmented lactone ring, and three of them (compounds 3-5) exhibited cytotoxicity against the human pancreatic cancer cell line MIA PaCa-2 with IC50 values ranging from 0.9 to 1.2 µg/mL. Genome sequencing and CRISPR/dCas9-assisted gene knockdown led to the identification of the ~43 kb balmoralmycin biosynthetic gene cluster (bal BGC). The bal BGC encodes a type II polyketide synthase (PKS) system for assembling the angucycline aglycone, six enzymes for generating the deoxysugar d-olivose, and a hybrid type II/III PKS system for synthesizing the 2,4-decadienoic acid chain. Based on the genetic and chemical information, we propose a mechanism for the biosynthesis of balmoralmycin and the shunt products. The chemical and genetic studies yielded insights into the biosynthetic origin of the structural diversity of angucyclines. IMPORTANCE Angucyclines are structurally diverse aromatic polyketides that have attracted considerable attention due to their potent bioactivity and intriguing biosynthetic origin. Balmoralmycin is a representative of a small family of angucyclines with unique structural features and an unknown biosynthetic origin. We report a newly isolated Streptomyces strain that produces balmoralmycin in a high fermentation titer as well as several structurally related shunt products. Based on the chemical and genetic information, a biosynthetic pathway that involves a type II polyketide synthase (PKS) system, cyclases/aromatases, oxidoreductases, and other ancillary enzymes was established. The elucidation of the balmoralmycin pathway enriches our understanding of how structural diversity is generated in angucyclines and opens the door for the production of balmoralmycin derivatives via pathway engineering.

CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932.

Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp. F6932 was isolated from white mangrove (Avicennia officinalis) in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C8 and a new analog palmarumycin CP30. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)-mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain.

Enaminone Formation Drives the Coupling of Biosynthetic Pathways to Generate Cyclic Lipopeptides.

A family of novel cyclic lipopeptides named tasikamides A-H (Tsk A-H) were discovered recently in Streptomyces tasikensis P46. Aside from the unique cyclic pentapeptide scaffold shared by the tasikamides, Tsk A-C contain a hydrazone bridge that connects the cyclic pentapeptide to the lipophilic alkyl 5-hydroxylanthranilate (AHA) moiety. Here we report the production of tasikamides I-K (Tsk I-K) by a mutant strain of S. tasikensis P46 that overexpresses two pathway-specific transcription regulators. Unlike Tsk A-C, Tsk I-K feature a rare enaminone-bridge that links the cyclic peptide scaffold to the AHA moiety. Our experimental data suggest that Tsk I-K are generated by the coupling of two biosynthetic pathways via a nonenzymatic condensation reaction between an arylamine and a ß-keto aldehyde-containing precursor. The results underscore the nucleophilic and electrophilic reactivity of the ß-keto aldehyde moiety and its ability to promote fragment coupling reactions in live microbial cells.

Phytochemical and biological studies on rare and endangered plants endemic to China. Part XXV. Structurally diverse triterpenoids and diterpenoids from two endangered Pinaceae plants endemic to the Chinese Qinling Mountains and their bioactivities.

A joint phytochemical investigation on the MeOH extracts of the twigs and needles of two endangered Pinaceae plants endemic to the Chinese Qinling Mountains, Picea neoveitchii (an evergreen spruce) and Larix potaninii var. chinensis (a deciduous larch), led to the isolation and characterization of 34 and 24 structurally diverse terpenoids, respectively. Among them, seven are previously undescribed, including a picane-type [i.e., 14(13 â†’ 12)abeo-12αH-serratane] (neoveitchin A) and a serratane-type (neoveitchin B) triterpenoids, and an abietane-type (neoveitchin C) as well as four labdane-type (potalarxins A-D) diterpenoids. Their structures and absolute configurations were established by extensive spectroscopic methods and/or X-ray diffraction analyses. All isolates were evaluated for their inhibitory activities against the human protein tyrosine phosphatase 1B (PTP1B). Serrat-14-en-3α,21ß-diol, betulinic acid, 3ß-hydroxy-11-ursen-13(28)-olide, ursolic acid, and oleanolic acid were found to have considerable inhibitory effects against PTP1B, with IC50 values ranging from 1.1 to 18.1 µM. The interactions of the bioactive triterpenoids with PTP1B were thereafter performed by employing molecular docking studies. In addition, 7-oxo-dehydroabietic acid (an abietane-type diterpenoid) and mangiferonic acid (a cycloartane-type triterpenoid) inhibited acetyl-coenzyme A carboxylase 1 (ACC1), with IC50 values of 3.4 and 6.6 µM, respectively.

Biosynthesis of Tasikamides via Pathway Coupling and Diazonium-Mediated Hydrazone Formation.

Naturally occurring hydrazones are rare despite the ubiquitous usage of synthetic hydrazones in the preparation of organic compounds and functional materials. In this study, we discovered a family of novel microbial metabolites (tasikamides) that share a unique cyclic pentapeptide scaffold. Surprisingly, tasikamides A-C (1-3) contain a hydrazone group (C═N─N) that joins the cyclic peptide scaffold to an alkyl 5-hydroxylanthranilate (AHA) moiety. We discovered that the biosynthesis of 1-3 requires two discrete gene clusters, with one encoding a nonribosomal peptide synthetase (NRPS) pathway for assembling the cyclic peptide scaffold and another encoding the AHA-synthesizing pathway. The AHA gene cluster encodes three ancillary enzymes that catalyze the diazotization of AHA to yield an aryl diazonium species (diazo-AHA). The electrophilic diazo-AHA undergoes nonenzymatic Japp-Klingemann coupling with a ß-keto aldehyde-containing cyclic peptide precursor to furnish the hydrazone group and yield 1-3. The studies together unraveled a novel mechanism whereby specialized metabolites are formed by the coupling of two biosynthetic pathways via an unprecedented in vivo Japp-Klingemann reaction. The findings raise the prospect of exploiting the arylamine-diazotizing enzymes (AAD) for the in vivo synthesis of aryl compounds and modification of biological macromolecules.

Structurally diverse glycosides of secoiridoid, bisiridoid, and triterpene-bisiridoid conjugates from the flower buds of two Caprifoliaceae plants and their ATP-citrate lyase inhibitory activities.

The ethanolic extracts of the dried flower buds of two Caprifoliaceae plants, Lonicera japonica and Abelia × grandiflora, showed considerable inhibitory activities against adenosine triphosphate (ATP)-citrate lyase (ACL), a new promising drug target for the treatment of metabolic disorders. Bioassay-guided purification in conjunction with HPLC-PDA profiling led to the isolation and characterization of thirty-five (1-35) and fourteen (1'-14') structurally diverse compounds from the above two plant extracts, respectively. Compounds 1-9 and 1'-6' are previously undescribed glycosides. Their structures were elucidated on the basis of spectroscopic data, electronic circular dichroism (ECD), and single crystal X-ray diffraction analyses. In particular, lonicejaposide A (1) has an unprecedented skeleton generated through the coupling of C-7 in secologanin with C-2'' in phenylacetaldehyde via an aldol condensation. Abeliflorosides A (1') and B (2') are hitherto unknown glycosides of triterpene and bisiridoid conjugates constructed through the formation of a 1,3-dioxane moiety. All the isolates were evaluated for their inhibitory activities against ACL. Compounds 9, 25-28, 31, 1', 2', and 14' displayed significant inhibitory effects, with IC50 values ranging from 0.1 to 14.2 µM. The interactions of selected compounds possessing different structure features (e.g., 9, 25, 31, and 2') with ACL were thereafter performed by employing molecular docking studies. In addition, compound 2', the most complex triterpene-bisiridoid conjugate glycoside reported herein, also inhibited acetyl-CoA carboxylase 1 (ACC1), with an IC50 value of 7.9 µM. The dried material of the flower buds of L. japonica (honeysuckle) is a well-known traditional oriental medicine (i.e., Flos Lonicerae Japonicae, FLJ) and has long been used in large quantities. The above findings not only provide new insights for the development of multipurpose utilization of FLJ in healthcare community, but also provide profitable clues indicating that the flower buds of A. × grandiflora might be a potential alternative to FLJ in the traditional Chinese medicine market.

Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes.

Anthraquinone-fused enediynes (AQEs) are renowned for their distinctive molecular architecture, reactive enediyne warhead, and potent anticancer activity. Although the first members of AQEs, i.e., dynemicins, were discovered three decades ago, how their nitrogen-containing carbon skeleton is synthesized by microbial producers remains largely a mystery. In this study, we showed that the recently discovered sungeidine pathway is a "degenerative" AQE pathway that contains upstream enzymes for AQE biosynthesis. Retrofitting the sungeidine pathway with genes from the dynemicin pathway not only restored the biosynthesis of the AQE skeleton but also produced a series of novel compounds likely as the cycloaromatized derivatives of chemically unstable biosynthetic intermediates. The results suggest a cascade of highly surprising biosynthetic steps leading to the formation of the anthraquinone moiety, the hallmark C8-C9 linkage via alkyl-aryl cross-coupling, and the characteristic epoxide functionality. The findings provide unprecedented insights into the biosynthesis of AQEs and pave the way for examining these intriguing biosynthetic enzymes.

Cytotoxic secondary metabolites from the vulnerable conifer Cephalotaxus oliveri and its associated endophytic fungus Alternaria alternate Y-4-2.

Rare and endangered plants (REPs) and their associated endophytes survived in unique habitats are promising sources for natural product-derived drug discovery. In this study, six new (cephaloverines A-F, 1-6, resp.) and 16 known (11-26) cephalotaxine-type alkaloids, together with three new (oliverbiflavones A-C, 7-9, resp.) and 11 known (27-37) biflavonoids were isolated and characterized from the twigs and leaves of Cephalotaxus oliveri, an endangered plant endemic to China. Meanwhile, a preliminary investigation on the secondary metabolites from a selected fungal endophyte (i.e., Alternaria alternate Y-4-2) associated with the title plant led to the isolation of 21 structurally distinct polyketides including one new dimeric xanthone (10). The new structures (1-10) with the absolute configurations were determined by detailed spectroscopic analyses, electronic circular dichroism (ECD) or Na2MoO4-induced ECD, the modified Mosher's method, and some chemical transformations. Compounds 1-4 are the first representatives of naturally occurring N-oxides of cephalotaxine esters, while compounds 7-9 have a special structural feature of having a C-methylated biflavonoid skeleton. The Cephalotaxus alkaloids with ester side-chains at C-3 (1-6, 13-22, and 26) and four biflavonoids (27-29 and 34) were found to show pronounced cytotoxicities against a small panel of human cancer cell lines (A549, NCI-H460, HL60, NCI-H929, and RPMI-8226), with IC50 values mainly ranging from 0.003 to 9.34 µM. The most potent compound, deoxyharringtonine (16), generally exhibited IC50 values less than 10 nM. The structure-activity relationship (SAR) of the aforementioned Cephalotaxus alkaloids was briefly discussed.

Taming the flagellar motor of pseudomonads with a nucleotide messenger.

Pseudomonads rely on the flagellar motor to rotate a polar flagellum for swimming and swarming, and to sense surfaces for initiating the motile-to-sessile transition to adopt a surface-dwelling lifestyle. Deciphering the function and regulation of the flagellar motor is of paramount importance for understanding the behaviours of environmental and pathogenic pseudomonads. Recent studies disclosed the preeminent role played by the messenger c-di-GMP in controlling the real-time performance of the flagellar motor in pseudomonads. The studies revealed that c-di-GMP controls the dynamic exchange of flagellar stator units to regulate motor torque/speed and modulates the frequency of flagellar motor switching via the chemosensory signalling pathways. Apart from being a rotary motor, the flagellar motor is emerging as a mechanosensor that transduces surface-induced mechanical signals into an increase of cellular c-di-GMP concentration to initiate the cellular programs required for long-term colonization. Collectively, the studies generate long-awaited mechanistic insights into how c-di-GMP regulates bacterial motility and the motile-to-sessile transition. The new findings also raise the fundamental questions of how cellular c-di-GMP concentrations are dynamically coupled to flagellar output and the proton-motive force, and how c-di-GMP signalling is coordinated spatiotemporally to fine-tune flagellar response and the behaviour of pseudomonads in solutions and on surfaces.

Sungeidines from a Non-canonical Enediyne Biosynthetic Pathway.

We report the genome-guided discovery of sungeidines, a class of microbial secondary metabolites with unique structural features. Despite evolutionary relationships with dynemicin-type enediynes, the sungeidines are produced by a biosynthetic gene cluster (BGC) that exhibits distinct differences from known enediyne BGCs. Our studies suggest that the sungeidines are assembled from two octaketide chains that are processed differently than those of the dynemicin-type enediynes. The biosynthesis also involves a unique activating sulfotransferase that promotes a dehydration reaction. The loss of genes, including a putative epoxidase gene, is likely to be the main cause of the divergence of the sungeidine pathway from other canonical enediyne pathways. The findings disclose the surprising evolvability of enediyne pathways and set the stage for characterizing the intriguing enzymatic steps in sungeidine biosynthesis.

Correction: Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin.

Correction for 'Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin' by Zhen Jie Low et al., Chem. Commun., 2020, DOI: .

Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin.

Produced by a newly isolated Streptomycetes strain, meijiemycin is a gigantic linear polyene-polyol that exhibits structural features not seen in other members of the polyene-polyol family. We propose a biosynthetic mechanism and demonstrate that meijiemycin inhibits hyphal growth by inducing the aggregation of ergosterol and restructuring of the fungal plasma membrane.

LC-MS guided isolation and dereplication of Lycopodium alkaloids from Lycopodium cernuum var. sikkimense of different geographical origins.

Lycopodium alkaloids (LAs) are the characteristic metabolites of club mosses. Chemical differences often exist in different specimens of a single plant species collected from different geographic origins. In this study, a preliminary LC-MS detection and dereplication analyses of alkaloidal constituents of Lycopodium cernuum var. sikkimense (Müll. Hal.) C.B. Clarke (LCVS2) collected from Fujian province led to the isolation and characterization of three undescribed LAs, lycocernuskines A-C, and six known cernuane-type LAs. The known compounds were previously isolated from the same plant species (LCVS1) collected from Chongqing, and so their dereplication in LCVS2 was accomplished based on their retention times (tR) and the quasi-molecular ion peaks in the LC-MS fingerprint. Chemical structures were identified by spectroscopic methods, single-crystal X-ray diffraction analysis, and electronic circular dichroism calculations. Lycocernuskines A and B are the first two examples of C-12 hydroxylated phlegmarane-type LAs bearing a nitrone residue at the quinoline ring. The isolates were evaluated for their anti-AChE and neuroprotective effects.

Structurally Diverse Sesquiterpenoids from the Endangered Ornamental Plant Michelia shiluensis.

A preliminary phytochemical investigation on the MeOH extract of the leaves and twigs of the endangered ornamental plant Michelia shiluensis led to the isolation of 16 sesquiterpenoids. The isolated compounds comprised germacrane- (1-4, 13, 14), guaiane- (5-9, 15), amorphane- (10), and eudesmane-type (11, 12, 16) sesquiterpenoids. The new structures (1-12) were elucidated by spectroscopic and computational methods, and their absolute configurations (except for 9) were assigned by single-crystal X-ray diffraction crystallographic data and/or electronic circular dichroism spectra. Shiluolides (A-D, 1-4) are unprecedented C16 or C17 homogermacranolides, and their putative biosynthetic pathways are briefly discussed. Shiluone D (8) is a rare 1,10- seco-guaiane sesquiterpenoid featuring a new ether-containing spirocyclic ring, whereas shiluone E (9) represents the first example of a 1,5-4,5-di- seco-guaiane with a rare 5,11 -lactone moiety. Shiluone F (10) is the first amorphane-type sesquiterpenoid possessing an oxetane ring bridging C-1 and C-7. Bioassay evaluations indicated that lipiferolide (13) showed noteworthy cytotoxicities toward human cancer cell lines MCF-7 and A-549, with IC50 values of 1.5 and 7.3 µM, respectively. Shiluone D (8) exerted inhibition against protein tyrosine phosphatase 1B (IC50: 46.3 µM).

LC-MS guided isolation of sinodamines A and B: Chimonanthine-type alkaloids from the endangered ornamental plant Sinocalycanthus chinensis.

Two previously undescribed chimonanthine-type [sinodamines A and B] and five related known dimeric tryptamine-derived alkaloids were isolated and characterized from the leaves of the endangered ornamental plant Sinocalycanthus chinensis under the guidance of LC-MS detection and dereplication analyses, along with conventional isolation procedures. Their structures were established on the basis of spectroscopic methods and chemical transformations. Sinodamine A can be regarded as the naturally occurring N-oxide derivative of its pseudo-mesomer sinodamine B. An acid-catalyzed Meisenheimer rearrangement from sinodamine A to its oxazine-form with a final equilibrium of 1:2 was observed by monitoring their NMR spectra. (-)-Folicanthine showed significant cytotoxicity against human lung carcinoma A549 and colorectal carcinoma HT29 cells, with IC50 values of 7.76 and 6.16 µM, respectively.