RESUMEN
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 10â mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples.
Asunto(s)
Productos Biológicos/química , Mezclas Complejas/química , Laurencia/química , Sesquiterpenos/química , Animales , Productos Biológicos/aislamiento & purificación , Mezclas Complejas/aislamiento & purificación , Cristalización/métodos , Estructura Molecular , Sesquiterpenos/aislamiento & purificaciónRESUMEN
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.
RESUMEN
The structure of fuliginone was revised from a phenyl substituted phenalenone to a hydroxyl substituted phenalenone as a result of its re-purification via HPLC with subsequent NMR analysis together with an independent synthesis and analysis of the crystal structure, which was secured via the crystalline sponge method. On-flow High Performance Liquid Chromatography coupled to Nuclear Magnetic Resonance spectroscopy (HPLC-NMR) was employed to confirm the presence of the natural product in the plant extract and to monitor for any possible degradation or conversion of the compound.
Asunto(s)
Fenalenos/química , Fenalenos/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificaciónRESUMEN
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 100â µg, and the majority (95â µg) could be recovered after the experiments.
Asunto(s)
Alquinos/química , Productos Biológicos/química , Furanos/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Cristalografía por Rayos X , Estructura MolecularRESUMEN
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 inâ vivo and inâ 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.
Asunto(s)
Sesterterpenos/biosíntesis , Transferasas Alquil y Aril/metabolismo , Aspergillus oryzae/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Cristalografía por Rayos X , Emericella/química , Emericella/metabolismo , Espectroscopía de Resonancia Magnética , Conformación Molecular , Sesterterpenos/química , EstereoisomerismoRESUMEN
An enantio-, diastereo-, regio-, and chemoselective phosphine-catalyzed ß,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.
RESUMEN
Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.
Asunto(s)
Proteínas Fúngicas/química , Hierro/química , Ácidos Cetoglutáricos/química , Oxidorreductasas N-Desmetilantes/química , Terpenos/química , Androsterona/química , Androsterona/metabolismo , Sitios de Unión , Biocatálisis , Cationes Bivalentes , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Humanos , Hidroxilación , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Modelos Moleculares , Mutación , Oxidación-Reducción , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Progesterona/química , Progesterona/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Especificidad por Sustrato , Terpenos/clasificación , Terpenos/metabolismo , Testosterona/química , Testosterona/metabolismoRESUMEN
The pyrimido[1,2-a][1,4]benzodiazepines (1a-c) and the 8-membered analogues (diazocines 2a and 2b) were separated into their atropisomers with HPLC on a chiral column. High stereochemical stability was observed in the atropisomer of the 8-membered derivatives (2a and 2b), and the 1,4-benzodiazepine (1c) with 2'-chloro at the pendant phenyl showed a lower energy barrier for the conversion between the atropisomers compared with that with the unsubstituted pendant phenyl (1a). The aR isomer of 1a-c was revealed to be the eutomer in GABA(A) receptor binding, and the eutomer 1c-R showed extremely potent activity with an IC(50) value of 1.5 nM.
Asunto(s)
Benzodiazepinas/química , Receptores de GABA-A/química , Ácido gamma-Aminobutírico/química , Benzodiazepinas/metabolismo , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Humanos , Estructura Molecular , Unión Proteica , Receptores de GABA-A/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Cycloelatanene A and B are marine natural products first reported a few years ago. Their relative structures had been elucidated by an extensive NMR study and found to be epimers. However, their absolute configurations had not been established because they were isolated in only minute quantities as oily compounds. In this study, the complete structures of cycloelatanene A and B, including absolute configurations, were determined by the crystalline sponge method. The structure analysis confirmed the unique tricyclic structure involving a spiro[5.5]undecene skeleton. One stereogenic centre at C4 was revised as a result of this analysis. Since it only took 1-2 weeks to complete the experiments using the crystalline sponge method (guest-soaking followed by crystallographic analysis), this method is now highly recommended as a first port of call to achieve complete natural product structure elucidation.
RESUMEN
Dibenzo[b,d]azepin-6-ones (2a,b) were separated by chiral HPLC into the aR- and aS-atropisomers with high stereochemical stability, and methylation at C7 of 2a stereoselectively gave the (aR*,7R*) isomer (4a), which converted to the thermodynamically stable (aS*,7R*) atropisomer (5a) after heating.
Asunto(s)
Azepinas/síntesis química , Benceno/química , Azepinas/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Metilación , Modelos Moleculares , Estructura Molecular , EstereoisomerismoRESUMEN
We designed and synthesized hydroxamic acid derivatives bearing a 4-(3-pyridyl)phenyl group as a cap structure, and found that they exhibit potent histone deacetylase (HDAC) inhibitory activity. A representative compound, 17a, showed more potent growth-inhibitory activity against pancreatic cancer cells and greater upregulation of p21(WAF1/CIP1) expression than the clinically used HDAC inhibitor suberoylanilide hydroxamic acid (Zolinza).