Biogenetic Precursor Research Articles

Efficient Production of Some Bioactive Depsides and Simple Phenolic Acids by Microshoots of Aronia × Prunifolia (Purple Aronia) Agitated Cultures as the Result of Feeding Strategy with Four Different Biogenetic Precursors

A precursor feeding strategy was used for the first time in agitated microshoot cultures of Aronia × prunifolia. This strategy involved the addition of biogenetic precursors of simple phenolic acids (phenylalanine, cinnamic acid, and benzoic acid) and depsides (caffeic acid) into the culture media, with an assessment of its effect on the production of these bioactive compounds. The in vitro cultures were maintained in Murashige–Skoog medium (1 mg/L BAP and 1 mg/L NAA). Precursors at five concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 mmol/L) were fed into the medium at the time of culture initiation (point “0”) and independently on the 10th day of growth cycles. The contents of 23 compounds were determined in methanolic extracts of biomass collected after 20 days of growth cycles using an HPLC method. All extracts contained the same four depsides (chlorogenic, neochlorogenic, rosmarinic, and cryptochlorogenic acids) and the same four simple phenolic acids (protocatechuic, vanillic, caffeic, and syringic acids). Chlorogenic and neochlorogenic acids were the predominant compounds in all extracts (max. 388.39 and 263.54 mg/100 g d.w.). The maximal total contents of all compounds were confirmed after feeding with cinnamic acid (5 mmol/L, point “0”) and caffeic acid (10 mmol/L, point “0”), which caused a 2.68-fold and 2.49-fold increase in the contents of the estimated compounds vs. control cultures (603.03 and 558.48 mg/100 g d.w., respectively). The obtained results documented the efficacy of the precursor feeding strategy in enhancing the production of bioactive compounds in agitated cultures of A. × prunifolia and suggest a potential practical application value.

Discovery of Unusual Ajmaline-Macroline Type Bisindole Alkaloids from Alstonia macrophylla by Building Blocks-Based Molecular Networking.

Three unusual ajmaline-macroline type bisindole alkaloids, alsmaphylines A-C, together with their postulated biogenetic precursors, were isolated from the stem barks and leaves of Alstonia macrophylla via the building blocks-based molecular network (BBMN) strategy. Alsmaphyline A represents a rare ajmaline-macroline type bisindole alkaloid with an S-shape polycyclic ring system. Alsmaphylines B and C are two novel ajmaline-macroline type bisindole alkaloids with N-1-C-21' linkages, and the former possesses an unconventional stacked conformation due to the presence of intramolecular noncovalent interactions. The chemical structures including absolute configurations of alsmaphylines A-C were established by comprehensive spectroscopic analyses, electronic circular dichroism (ECD) calculations, and single-crystal X-ray crystallography. In addition, a plausible biosynthetic pathway of these bisindole alkaloids as well as their ability to promote the protein synthesis on HT22 cells were discussed.

Adenophorone, An Unprecedented Sesquiterpene from Eupatorium adenophorum: Structural Elucidation, Bioinspired Total Synthesis and Neuroprotective Activity Evaluation

Abstract(−)‐Adenophorone (1), a caged polycyclic sesquiterpene featuring an unprecedented tricyclo[4.3.1.05,9]decane skeleton, was isolated from Eupatorium adenopharum Spreng. The structure of 1 was unambiguously established by a combination of spectroscopic analysis, X‐ray crystallography, and bioinspired total synthesis. Key synthetic features include a sequential Reformatsky/oxidation/regio‐ and stereoselective hydrogenation, and subsequent merged MBH–Tsuji–Trost cyclization. The concise synthetic sequence efficiently constructs the bicyclic skeleton of cadinene sesquiterpene (+)‐euptox A (2) in 8 steps from commercially available monoterpene (−)‐carvone (6), with outstanding performance on diastereocontrol. The bioinspired synthesis of 1 was achieved from 2, a plausible biogenetic precursor, via transannular Michael addition. This work provides experimental evidence of our proposed biosynthetic hypothesis of 1. Additionally, compound 1 showed potent neuroprotective activity in H2O2‐treated SH‐SY5Y and PC12 cells.

Adenophorone, An Unprecedented Sesquiterpene from Eupatorium adenophorum: Structural Elucidation, Bioinspired Total Synthesis and Neuroprotective Activity Evaluation.

(-)-Adenophorone (1), a caged polycyclic sesquiterpene featuring an unprecedented tricyclo[4.3.1.05,9 ]decane skeleton, was isolated from Eupatorium adenopharum Spreng. The structure of 1 was unambiguously established by a combination of spectroscopic analysis, X-ray crystallography, and bioinspired total synthesis. Key synthetic features include a sequential Reformatsky/oxidation/regio- and stereoselective hydrogenation, and subsequent merged MBH-Tsuji-Trost cyclization. The concise synthetic sequence efficiently constructs the bicyclic skeleton of cadinene sesquiterpene (+)-euptox A (2) in 8 steps from commercially available monoterpene (-)-carvone (6), with outstanding performance on diastereocontrol. The bioinspired synthesis of 1 was achieved from 2, a plausible biogenetic precursor, via transannular Michael addition. This work provides experimental evidence of our proposed biosynthetic hypothesis of 1. Additionally, compound 1 showed potent neuroprotective activity in H2 O2 -treated SH-SY5Y and PC12 cells.

Phyllospongianes A-E, Dinorscalarane Sesterterpenes from the Marine Sponge Phyllospongia foliascens.

Phyllospongianes A-E (1-5), five new scalarane derivatives featuring an unprecedented 6/6/6/5 tetracyclic dinorscalarane scaffold, along with the known probable biogenetic precursor, 12-deacetylscalaradial (6), were isolated from the marine sponge Phyllospongia foliascens. The structures of the isolated compounds were determined by analysis of spectroscopic data and electronic circular dichroism experiments. Compounds 1-5 are the first 6/6/6/5 tetracyclic scalarane derivatives to be reported within the scalarane family. Compounds 1, 2, and 4 exhibited antibacterial activity against Vibrio vulnificus, Vibrio parahemolyticus, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, and Pseudomonas aeruginosa with MIC values ranging from 1 to 8 μg/mL. Furthermore, compound 3 exhibited significant cytotoxic activity on MDA-MB-231, HepG2, C4-2-ENZ, MCF-7, H460, and HT-29 cancer cell lines with IC50 values in the range between 0.7 and 13.2 μM.

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.

Monoterpenoid indole alkaloid adducts and dimers from Melodinus fusiformis

Eight unprecedented monoterpenoid indole alkaloid (MIA) adducts and dimers, melofusinines A–H (1–8), and three undescribed melodinus-type MIA monomers, melofusinines I–K (9–11), together with six putative biogenetic precursors were isolated from the twigs and leaves of Melodinus fusiformis Champ. ex Benth. Compounds 1 and 2 are unusual hybrid indole alkaloids incorporating an aspidospermatan-type MIA with a monoterpenoid alkaloid unit via C–C coupling. Compounds 3–8 feature the first MIA dimers constructed through an aspidospermatan-type monomer and a rearranged melodinus-type monomer with two different types of couplings. Their structures were elucidated by spectroscopic data, single crystal X-ray diffraction, and calculated electric circular dichroism spectra analysis. In addition, dimers 5 and 8 showed significant neuroprotection effects on MPP +-injured primary cortical neurons.

Late-Stage C(sp2 )-H Arylation of Artemisinic Acid and Arteannuin B: Effect of Olefin Migration Towards Synthesis of C-13 Arylated Artemisinin Derivatives.

In recent years, C-H bond functionalization has emerged as a pivotal tool for late-stage functionalization of complex natural products for the synthesis of potent biologically active derivatives. Artemisinin and its C-12 functionalized semi-synthetic derivatives are well-known clinically used anti-malarial drugs due to the presence of the essential 1,2,4-trioxane pharmacophore. However, in the wake of parasite developing resistance against artemisinin-based drugs, we conceptualized the synthesis of C-13 functionalized artemisinin derivatives as new antimalarials. In this regard, we envisaged that artemisinic acid could be a suitable precursor for the synthesis of C-13 functionalized artemisinin derivatives. Herein, we report C-13 arylation of artemisinic acid, a sesquiterpene acid and our attempts towards synthesis of C-13 arylated artemisinin derivatives. However, all our efforts resulted in the formation of a novel ring-contracted rearranged product. Additionally, we have extended our developed protocol for C-13 arylation of arteannuin B, a sesquiterpene lactone epoxide considered to be the biogenetic precursor of artemisinic acid. Indeed, the synthesis of C-13 arylated arteannuin B renders our developed protocol to be effective in sesquiterpene lactone as well.

Different Types of Hypericum perforatum cvs. (Elixir, Helos, Topas) In Vitro Cultures: A Rich Source of Bioactive Metabolites and Biological Activities of Biomass Extracts

Microshoot agitated and bioreactor cultures (PlantForm bioreactors) of three Hypericum perforatum cultivars (Elixir, Helos, Topas) were maintained in four variants of Murashige and Skoog medium (MS) supplemented with 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (in the range of 0.1-3.0 mg/L). In both types of in vitro cultures, the accumulation dynamics of phenolic acids, flavonoids, and catechins were investigated during 5- and 4-week growth cycles, respectively. The contents of metabolites in methanolic extracts from biomasses collected in 1-week intervals were estimated by HPLC. The highest total contents of phenolic acids, flavonoids, and catechins were 505, 2386, and 712 mg/100 g DW, respectively (agitated cultures of cv. Helos). The extracts from biomass grown under the best in vitro culture conditions were examined for antioxidant and antimicrobial activities. The extracts showed high or moderate antioxidant activity (DPPH, reducing power, and chelating activity assays), high activity against Gram-positive bacteria, and strong antifungal activity. Additionally, experiments with phenylalanine feeding (1 g/L) in agitated cultures were performed reaching the highest enhancement of the total contents of flavonoids, phenolic acids, and catechins on day 7 after the addition of the biogenetic precursor (2.33-, 1.73- and 1.33-fold, respectively). After feeding, the highest accumulation of polyphenols was detected in the agitated culture of cv. Elixir (4.48 g/100 g DW). The high contents of metabolites and the promising biological properties of the biomass extracts are interesting from a practical point of view.

Identification of Unstable Ellagitannin Metabolites in the Leaves of Quercus dentata by Chemical Derivatization.

The identification of unstable metabolites of ellagitannins having ortho-quinone structures or reactive carbonyl groups is important to clarify the biosynthesis and degradation of ellagitannins. Our previous studies on the degradation of vescalagin, a major ellagitannin of oak young leaves, suggested that the initial step of the degradation is regioselective oxidation to generate a putative quinone intermediate. However, this intermediate has not been identified yet. In this study, young leaves of Quercus dentata were extracted with 80% acetonitrile containing 1,2-phenylenediamine to trap unstable ortho-quinone metabolites, and subsequent chromatographic separation afforded a phenazine derivative of the elusive quinone intermediate of vescalagin. In addition, phenylenediamine adducts of liquidambin and dehydroascorbic acid were obtained, which is significant because liquidambin is a possible biogenetic precursor of C-glycosidic ellagitannins and ascorbic acid participates in the production of another C-glycosidic ellagitannin in matured oak leaves.

Chemical Emulation of the Biosynthetic Route to Anthrasteroids: Synthesis of Asperfloketal A.

The anthrasteroid rearrangement has been discussed for the formation of the eponymous substance class since its discovery. We here report its chemical emulation from a plausible biogenetic precursor and show how it accounts for the formation of asperfloketals A and B through a mechanistic bifurcation event. As a result, these natural products arise from double Wagner-Meerwein rearrangements and, thus, are 1(10→5),1(5→6)- and 1(10→5),4(5→6)diabeo-14,15-secosteroids, respectively. To establish an efficient route to a bioinspired precursor, we devised a sequence of orchestrated oxidative activation and rearrangement from ergosterol.

Cultivation of Inonotus hispidus in Stirred Tank and Wave Bag Bioreactors to Produce the Natural Colorant Hispidin

Hispidin (6-(3,4-dihydroxystyrl)-4-hydroxy-2-pyrone) production in submerged cultured mycelia of the basidiomycete Inonotus hispidus was doubled in shake flasks through irradiation with white light. The daily addition of 1 mM hydrogen peroxide as a chemical stressor and a repeated supplementation of the shake flask cultures with 2 mM caffeic acid, a biogenetic precursor, further increased the hispidin synthesis. These cultivation conditions were combined and applied to parallel fermentation trials on the 4 L scale using a classical stirred tank bioreactor and a wave bag bioreactor. No significant differences in biomass yield and colorant production were observed. The hispidin concentration in both bioreactors reached 5.5 g·L−1, the highest ever published. Textile dyeing with hispidin was successful, but impeded by its limited light stability in comparison to industrial dyes. However, following the idea of sustainability and the flawless toxicity profile, applications in natural cosmetics, other daily implements, or even therapeutics appear promising.

Phonerilins A–K, cytotoxic ingenane and ingol diterpenoids from Euphorbia neriifolia

A phytochemical study of the aerial parts of Euphorbia neriifolia led to the isolation of six new ingenane-type diterpenoids, phonerilins A–F (1–6), five new ingol-type diterpenoids, phonerilins G–K (7–11), and five known analogues (12–16). Their structures with absolute configurations were elucidated by a combined method. Phonerilin A (1) is the second rearranged ingenane diterpenoid with a unique 6/6/7/3-fused ring system. Based on single-crystal X-ray diffraction and NOESY data, the structure of 20-deoxy-16-hydroxyingenol was revised as 20-deoxy-17-hydroxyingenol (12) that is likely the biogenetic precursor of 1. Biological evaluation revealed some isolates exhibited cytotoxic effects against two human tumor cell lines, A549 and HL-60.

On the Erosion of Enantiopurity of Rhodonoids via Their Asymmetric Total Synthesis.

Rhodonoid natural products are found in nature as a scalemic mixture. This interesting phytochemical feature is presumed to originate from a reversible electrocyclic ring opening of the chromene core present in the biogenetic precursors of rhodonoids. Herein, we systematically investigated factors that are responsible for this racemization event. This eventually led us to complete the asymmetric total synthesis of rhodonoids A, C, D, and G.

Antioxidant Potential and Enhancement of Bioactive Metabolite Production in In Vitro Cultures of Scutellaria lateriflora L. by Biotechnological Methods.

Studies carried out using three different in vitro assays and a biological setting (Escherichia coil) demonstrated the antioxidant activity of Scutellaria lateriflora microshoot extract. Moreover, the extract exhibited no toxicity in a brine shrimp lethality bioassay. These results indicated that microshoots are a rich, safe source of antioxidants, which encouraged us to enhance their production in vitro. In agar and agitated cultures, two biotechnological strategies were applied: feeding the cultures with the biogenetic precursors of the phenolics—phenylalanine and tyrosine, and eliciting them with methyl jasmonate. Specific Scutellaria flavonoids and verbascoside were analysed by HPLC. Feeding with precursors (1 g/L) in agar cultures decreased the production of the metabolites. In agitated cultures, different concentrations of precursors (1.0–2.5 g/L) and the elicitor (10; 50; 100 µM) were tested. Additionally, parallel feeding with the precursor and elicitor in a concentration of 50 µM were applied. The best strategy for total flavonoid and verbascoside production was phenylalanine feeding (1.5 g/L), max. 3765 and 475 mg/100 g DW, respectively, after 7 days. This is the first report documenting the high antioxidant production in S. lateriflora microshoots after feeding with phenylalanine. Moreover, for the first time, bioreactor cultures were successfully maintained, obtaining attractive results (max. total flavonoid content 2348 and verbascoside 485 mg/100 g DW).

Stereocontrolled Synthesis and Structural Revision of Plebeianiol A.

We report the structural revision via synthesis of the abietane diterpenoid plebeianiol A. The synthesis was accomplished by a short and convergent sequence that featured our previously established cobalt-catalyzed hydrogen-atom-transfer-induced radical bicyclization. We further connected plebeianiol A as the likely biogenetic precursor to another previously reported ether-bridged abietane. Finally, we demonstrated that the key cyclization event is efficient with the A-ring diol protected as two different cyclic acetals or in unprotected form.

Secoiridoid dimers and their biogenetic precursors from the fruits of Cornus officinalis with potential therapeutic effects on type 2 diabetes

Cornusdiridoid A-F (1–6), six unusual cornuside-morroniside secoiridoid dimers, and their possible new biogenetic precursor, 3″,5″-dehydroxycornuside (7), together with four known secoiridoids (8–11), were obtained from the fruits of Cornus officinalis. Their structures were elucidated on the basis of various spectroscopic and chemical methods. A plausible biosynthetic pathway of compounds 1–11 was proposed. The α-glucosidase inhibitory, antioxidant and anti-inflammatory activities of these isolates were evaluated. Some of them emerged out as potent antidiabetic, anti-inflammatory and free radical scavenging agents. Molecular docking was also carried out for antidiabetic target α-glucosidase to investigate the possible binding modes of the most potent α-glucosidase inhibitor, vincosamide (9). These results revealed that the secoiridoids from C. officinalis fruits may be served as new potential antidiabetic agents to prevent and treat type 2 diabetes.

Bioinspired Network Analysis Enabled Divergent Syntheses and Structure Revision of Pentacyclic Cytochalasans.

We accomplished the divergent total syntheses of ten pentacyclic cytochalasans (aspergillin PZ, trichodermone, trichoderones, flavipesines, and flavichalasines) from a common precursor aspochalasin D and revised the structures of trichoderone B, spicochalasin A, flavichalasine C, aspergilluchalasin based on structure network analysis of the cytochalasans biosynthetic pathways and DFT calculations. The key steps of the syntheses include transannular alkene/epoxyalkene and carbonyl-ene cyclizations to establish the C/D ring of pentacyclic aspochalasans. Our bioinspired approach to these pentacyclic cytochalasans validate the proposed biosynthetic speculation from a chemical view and provide a platform for the synthesis of more than 400 valuable cytochalasans bearing different macrocycles and amino-acid residues.

Densazalin, a New Cytotoxic Diazatricyclic Alkaloid from the Marine Sponge Haliclona densaspicula.

Densazalin, a polycyclic alkaloid, was isolated from the marine sponge Haliclona densaspicula collected in Korea. The complete structure of the compound was determined by spectroscopic methods, including 1D and 2D nuclear magnetic resonance techniques, high-resolution mass spectrometry, and comparison of the calculated and measured electronic circular dichroism spectra. Densazalin possesses a unique 5,11-diazatricyclo[7.3.1.02,7]tridecan-2,4,6-triene moiety, which is connected by two linear carbon chains. This compound was derived from the biogenetic precursor bis-1,3-dialkylpyridnium. Densazalin exhibited cytotoxic activity on two human tumor cell lines (AGS and HepG2) in the Cell Counting Kit-8 (CCK-8) bioassay, with IC50 values ranging from 15.5 to 18.4 μM.

Total Synthesis of Homo- and Heterodimeric Bispyrrolidinoindoline Dioxopiperazine Natural Products.

Total synthesis and structural confirmation of homo- and heterodimeric bispyrrolidinoindoline dioxopiperazine alkaloids isolated from fungi and bacteria, namely, ditryptoleucine A, ditryptoleucine B (11), the N,N'-bis-demethylated analogue (+)-12, (-)-dibrevianamide F (13), (-)-SF-5280-451 (14), tetratryptomycin A (15), (-)-tryprophenaline (17), and (-)-SF-5280-415 (18), has been carried out starting from the corresponding bispyrrolidinoindolines derived from tryptophan. Our efforts to synthesize all possible diastereomers of the natural ditryptoleucine isolates uncovered structural factors that determine the rate and efficiency of dioxopiperazine ring formation, leading in some cases to mixtures of diastereomers by concomitant epimerization, to the formation of their putative monomeric dioxopiperazine dipeptide biogenetic precursors, and to the alternative formation of a dimer with a fused 1,3,5-triazepan-6-one heterocycle.