Sporopollenin-inspired design and synthesis of robust polymeric materials.

Sporopollenin is a mechanically robust and chemically inert biopolymer that constitutes the outer protective exine layer of plant spores and pollen grains. Recent investigation of the molecular structure of pine sporopollenin revealed unique monomeric units and inter-unit linkages distinct from other previously known biopolymers, which could be harnessed for new material design. Herein, we report the bioinspired synthesis of a series of sporopollenin analogues. This exercise confirms large portions of our previously proposed pine sporopollenin structural model, while the measured chemical, thermal, and mechanical properties of the synthetic sporopollenins constitute favorable attributes of a new kind of robust material. This study explores a new design framework of robust materials inspired by natural sporopollenins, and provides insights and reagents for future elucidation and engineering of sporopollenin biosynthesis in plants.

The chloroalkaloid (-)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants.

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.

New phenolic glycosides from Polygonum cuspidatum.

Two new isobenzofuranone derivatives, polyphthaliside A (1) and polyphthaliside B (2), and a new isocoumarin derivative, polyisocoumarin (3), were isolated from Polygonum cuspidatum. Their structures were elucidated by detailed spectroscopic analysis and chemical methods. The cytotoxicity activity and PTP1B inhibitory activity of compounds 1-3 were estimated and none of them exhibited activities at a concentration of 10 µM.

Exploration of icariin analog structure space reveals key features driving potent inhibition of human phosphodiesterase-5.

The natural product icariin inhibits human phosphodiesterase-5 (PDE5) and represents a unique pharmacophore for treating erectile dysfunction, pulmonary hypertension, and other diseases. In this study, we explore the available icariin-derived chemical scaffolds through medicinal chemistry to develop novel icariin PDE5 inhibitors with improved potency and specificity. We synthesized six novel semi-synthetic icariin analogs as well as three naturally occurring icariin analogs, and characterized the structure-activity relationship in the context of human PDE5 inhibition using in vitro enzyme inhibition and kinetics assays and molecular modeling. Mammalian-cell-based assays and in vitro enzyme inhibition assays against human PDE6C further helped to identify the most potent and selective icariin analogs. Our results reveal the synergistic contribution of functional groups at the C3 and C7 positions of the icariin backbone towards PDE5 inhibition. Whereas a hydrophobic and flexible alkanol group at the C7 position is sufficient to enhance icariin analog potency, combining this group with a hydrophilic sugar group at the C3 position leads to further enhancement of potency and promotes specificity towards PDE5 versus PDE6C. In particular, compounds 3 and 7 exhibit Ki values of 0.036 ± 0.005 µM and 0.036 ± 0.007 µM towards PDE5 respectively, which are approaching those of commercial PDE5 inhibitors, and can effectively reduce GMP levels in cultured human BJ-hTERT cells. This study identifies novel icariin analogs as potent and selective PDE5 inhibitors poised to become lead compounds for further pharmaceutical development.

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum-graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts.

The molecular structure of plant sporopollenin.

Sporopollenin is a ubiquitous and extremely chemically inert biopolymer that constitutes the outer wall of all land-plant spores and pollen grains1. Sporopollenin protects the vulnerable plant gametes against a wide range of environmental assaults, and is considered a prerequisite for the migration of early plants onto land2. Despite its importance, the chemical structure of plant sporopollenin has remained elusive1. Using a newly developed thioacidolysis degradative method together with state-of-the-art solid-state NMR techniques, we determined the detailed molecular structure of pine sporopollenin. We show that pine sporopollenin is primarily composed of aliphatic-polyketide-derived polyvinyl alcohol units and 7-O-p-coumaroylated C16 aliphatic units, crosslinked through a distinctive dioxane moiety featuring an acetal. Naringenin was also identified as a minor component of pine sporopollenin. This discovery answers the long-standing question about the chemical make-up of plant sporopollenin, laying the foundation for future investigations of sporopollenin biosynthesis and for the design of new biomimetic polymers with desirable inert properties.

Complete Pathway Elucidation and Heterologous Reconstitution of Rhodiola Salidroside Biosynthesis.

Salidroside is a bioactive tyrosine-derived phenolic natural product found in medicinal plants under the Rhodiola genus. In addition to their anti-fatigue and anti-anoxia roles in traditional medicine, Rhodiola total extract and salidroside have also displayed medicinal properties as anti-cardiovascular diseases and anti-cancer agents. The resulting surge in global demand of Rhodiola plants and salidroside has driven some species close to extinction. Here, we report the full elucidation of the Rhodiola salidroside biosynthetic pathway utilizing the first comprehensive transcriptomics and metabolomics datasets for Rhodiola rosea. Unlike the previously proposed pathway involving separate decarboxylation and deamination enzymatic steps from tyrosine to the key intermediate 4-hydroxyphenylacetaldehyde (4-HPAA), Rhodiola contains a pyridoxal phosphate-dependent 4-HPAA synthase that directly converts tyrosine to 4-HPAA. We further identified genes encoding the subsequent 4-HPAA reductase and tyrosol:UDP-glucose 8-O-glucosyltransferase, respectively, to complete salidroside biosynthesis in Rhodiola. We show that heterologous production of salidroside can be achieved in the yeast Saccharomyces cerevisiae as well as the plant Nicotiana benthamiana through transgenic expression of Rhodiola salidroside biosynthetic genes. This study provides new tools for engineering sustainable production of salidroside in heterologous hosts.

Demystifying traditional herbal medicine with modern approach.

Plants have long been recognized for their therapeutic properties. For centuries, indigenous cultures around the world have used traditional herbal medicine to treat a myriad of maladies. By contrast, the rise of the modern pharmaceutical industry in the past century has been based on exploiting individual active compounds with precise modes of action. This surge has yielded highly effective drugs that are widely used in the clinic, including many plant natural products and analogues derived from these products, but has fallen short of delivering effective cures for complex human diseases with complicated causes, such as cancer, diabetes, autoimmune disorders and degenerative diseases. While the plant kingdom continues to serve as an important source for chemical entities supporting drug discovery, the rich traditions of herbal medicine developed by trial and error on human subjects over thousands of years contain invaluable biomedical information just waiting to be uncovered using modern scientific approaches. Here we provide an evolutionary and historical perspective on why plants are of particular significance as medicines for humans. We highlight several plant natural products that are either in the clinic or currently under active research and clinical development, with particular emphasis on their mechanisms of action. Recent efforts in developing modern multi-herb prescriptions through rigorous molecular-level investigations and standardized clinical trials are also discussed. Emerging technologies, such as genomics and synthetic biology, are enabling new ways for discovering and utilizing the medicinal properties of plants. We are entering an exciting era where the ancient wisdom distilled into the world's traditional herbal medicines can be reinterpreted and exploited through the lens of modern science.

SG2-Type R2R3-MYB Transcription Factor MYB15 Controls Defense-Induced Lignification and Basal Immunity in Arabidopsis.

Lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response. However, the genetic pathway controlling defense-induced lignification remains unknown. Here, we demonstrate the Arabidopsis thaliana SG2-type R2R3-MYB transcription factor MYB15 as a regulator of defense-induced lignification and basal immunity. Loss of MYB15 reduces the content but not the composition of defense-induced lignin, whereas constitutive expression of MYB15 increases lignin content independently of immune activation. Comparative transcriptional and metabolomics analyses implicate MYB15 as necessary for the defense-induced synthesis of guaiacyl lignin and the basal synthesis of the coumarin metabolite scopoletin. MYB15 directly binds to the secondary wall MYB-responsive element consensus sequence, which encompasses the AC elements, to drive lignification. The myb15 and lignin biosynthetic mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with defense-induced lignin having a major role in basal immunity. A scopoletin biosynthetic mutant also shows increased susceptibility independently of immune activation, consistent with a role in preformed defense. Our results support a role for phenylalanine-derived small molecules in preformed and inducible Arabidopsis defense, a role previously dominated by tryptophan-derived small molecules. Understanding the regulatory network linking lignin biosynthesis to plant growth and defense will help lignin engineering efforts to improve the production of biofuels and aromatic industrial products as well as increase disease resistance in energy and agricultural crops.

Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate.

Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Synthase, upstream of the promiscuous oxidation step, acts as a practical method for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5α-ol, when coexpressed with CYP725A4. This works lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors.

Sulfoluciferin is Biosynthesized by a Specialized Luciferin Sulfotransferase in Fireflies.

Firefly luciferin is a specialized metabolite restricted to fireflies (family Lampyridae) and other select families of beetles (order Coleoptera). Firefly luciferin undergoes luciferase-catalyzed oxidation to produce light, thereby enabling the luminous mating signals essential for reproductive success in most bioluminescent beetles. Although firefly luciferin and luciferase have become widely used biotechnological tools, questions remain regarding the physiology and biochemistry of firefly bioluminescence. Here we report sulfoluciferin to be an in vivo derivative of firefly luciferin in fireflies and report the cloning of luciferin sulfotransferase (LST) from the North American firefly Photinus pyralis. LST catalyzes the production of sulfoluciferin from firefly luciferin and the sulfo-donor PAPS. Sulfoluciferin is abundant in several surveyed firefly genera as well as in the bioluminescent elaterid beetle Pyrophorus luminosus at a low level. We propose that sulfoluciferin could serve as a luciferin storage molecule in fireflies and that LST may find use as a new tool to modulate existing biotechnological applications of the firefly bioluminescent system.

Involvenflavones A-F, six new flavonoids with 3'-aryl substituent from Selaginella involven.

Six new flavonoids, involvenflavones A-F (1-6), were isolated from Selaginella involven. Their structures were elucidated based on UV, IR, 1D and 2D NMR as well as HR-ESI-MS techniques. All compounds belong to apigenin derivatives with 3'-aryl substituent. This is the first report of the apigenin derivatives with 3'-aryl substituent from nature resources. These compounds also exhibited a potent effect against the injury of human umbilical vein endothelial cell (HUVECs) induced by high concentrations of glucose in vitro.

New Selaginellin derivatives from Selaginella tamariscina.

Two new selaginellin derivatives selaginellin P (1) and selaginellin Q (2) were isolated from Selaginella tamariscina. The structures of 1 and 2 were established as 2,4'-dihydroxy-4-methyl-3-[(4-hydroxyphenyl)ethynyl]biphene (1) and 2,4'-dihydroxy-3-[(4-hydroxyphenyl)ethynyl]biphene (2) on the basis of spectroscopic means including HR-ESI-MS, 1D, and 2D NMR experiments.

Neuroprotective naphthalene and flavan derivatives from Polygonum cuspidatum.

Ten naphthalene derivatives including two unusual glycosides possessing a naphthalene-fused piceid via a [C8'-O-C6-C5-C7']-trans-dihydrofuran ring, two flavan derivatives, as well as sixteen known phenolic compounds, were isolated from Polygonum cuspidatum. The structures were determined by extensive NMR, MS, CD data, and chemical evidence. In the in vitro neuroprotective assays, at the concentration of 10 µM, five of these compounds exhibited significant effects against PC12 cells injured by rotenone.

Unciflavones A-F, six novel flavonoids from Selaginella uncinata (Desv.) Spring.

Six new flavonoids, unciflavones A-F (1-6), have been isolated from medicinal plant Selaginella uncinata (Desv.) Spring. Their structures were established on the basis of extensive NMR analysis including 1D NMR ((1)H, (13)C and DEPT) and 2D NMR (COSY, HSQC, HMBC) experiments as well as HRESIMS analysis. All compounds possess exceptional structural features with an aryl substituent at the C-8 position, which are uncommonly encountered in natural resources and firstly reported in genus Selaginella.

Uncinataflavone, a new flavonoid with a methyl benzoate substituent from Selaginella uncinata.

Uncinataflavone (1), a new flavonoid, together with four known compounds (2-5), was isolated from Selaginella uncinata (Desv.) Spring. Compounds 2 and 3 were isolated from the genus selaginella for the first time. The structure of the new compound was determined as methyl 3-(5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-6-yl)-4-methoxybenzoate by means of spectroscopic evidence, including UV, IR, 1D and 2D NMR analyses as well as HR-ESI-MS. These compounds (1-5) were evaluated for the antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl assay system.

A new flavonoid with 6-phenyl substituent from Selaginella uncinata.

A new flavonoid, 6-(5-acetyl-2-methoxyphenyl)-apigenin (1), together with nine known compounds (2-10), was isolated from Selaginella uncinata (Desv.) Spring. This is the first report of the spectroscopic data of compound 3. Compound 2 was first reported from this species. The structure of the new compound was elucidated on the basis of spectroscopic evidence, including 1D and 2D NMR as well as HR-EI-MS analysis.

Selaginellin M, a new selaginellin derivative from Selaginella pulvinata.

A new selaginellin derivative, selaginellin M (1), together with one known compound, selaginellin E (2), was isolated from Selaginella pulvinata. The structure of the new compound was elucidated and named as (R,S)-4-((4'-hydroxy-4-((2-hydroxyethoxy)methyl))-3-((4-hydroxyphenyl)ethynyl)biphenyl-2-yl)(4-hydroxyphenyl)methylene)cyclohexa-2,5-dienone on the basis of the spectroscopic data including UV, IR, 1D, and 2D NMR as well as HR-ESI-MS analysis.

Two new selaginellin derivatives from Selaginella tamariscina (Beauv.) Spring.

Two new selaginellin derivatives, selaginellins K (1) and L (2), were isolated from Selaginella tamariscina (Beauv.) Spring and characterized as 2-formyl-4,4'-dihydroxy-3-[(4-hydroxyphenyl)ethynyl]biphene and 4,4'-dihydroxy-2-methyl-3-[(4-hydroxyphenyl)ethynyl]biphene on the basis of their spectroscopic data including UV, IR, 1D, and 2D NMR as well as HR-ESI-MS spectroscopic analysis.

Selaginellins I and J, two new alkynyl phenols, from Selaginella tamariscina (Beauv.) Spring.

Selaginellins I (1) and J (2), two new compounds, were isolated from Selaginella tamariscina (Beauv.) Spring and were characterized as (R,S)-4-((2',4'-dihydroxy-4-(hydroxymethyl)-3-((4-hydroxyphenyl)ethynyl)biphenyl-2-yl)(4-hydroxyphenyl)methylene)cyclohexa-2,5-dienone (1) and (R,S)-4-((3-((3,4-dihydroxyphenyl)ethynyl)-4'-hydroxy-4-(hydroxymethyl)biphenyl-2-yl)(4-hydroxyphenyl)methylene)cyclohexa-2,5-dienone (2) on the basis of UV, IR, 1D and 2D NMR, and HR-ESI-MS spectroscopic analysis.