Stability of metal ion complexes with the synthetic phytosiderophore proline-2'-deoxymugineic acid.

Adequate micronutrient concentrations in crops are essential for human health and agricultural productivity. However, 30% of plants growing on cultivated soils worldwide are deficient in iron (Fe). Because of low micronutrient bioavailability, graminaceous plants have evolved to exude small molecules, called phytosiderophores, into the soil environment, which strongly complex and promote uptake of trace elements. The development of a synthetic phytosiderophore, proline-2'-deoxymugeneic acid (PDMA), has been shown to promote Fe uptake in rice plants; however, its binding capabilities with other metals, which may impact the ability to promote the uptake of Fe and other trace nutrient metals commonly found in soils, remain unknown. We conducted spectrophotometric titrations to determine the stability constants (logK) of PDMA complexes with Mn(II), Co(II), Cu(II), Ni(II), and Zn(II). We determined that PDMA complex stability constants correlated with: (1) the hydrolysis constants of metal ions (logKOH) in complexes; (2) the ionic potential of complexed metals; and (3) the corresponding complex stability constants of other mugineic acid type phytosiderophores, as well as the trishydroxamate microbial siderophore DFOB. These correlations demonstrate the potential, and limitations, on our ability to predict the stability of phytosiderophore complexes with metal ions with different physicochemical properties and with potentially different coordination structures.

Epoxide Ring-Opening Reactions for Abundant Production of Mugineic Acids and Nicotianamine Probes.

A succinct synthetic approach to mugineic acids and 2'-hydroxynicotianamine was established. Unlike all other synthetic methods, this approach utilized epoxide ring-opening reactions to form two C-N bonds and is characterized by the absence of redox reactions. Mugineic acid was synthesized from three readily available fragments on a gram scale in 6 steps. The protected 2'-hydroxynicotianamine was also synthesized in 4 steps, and the dansyl group, serving as a fluorophore, was introduced through a click reaction after propargylation of the 2'-hydroxy group. The dansyl-labeled nicotianamine (NA) iron complexes were internalized by oocytes overexpressing ZmYS1 (from maize) or PAT1 (from human) transporters, indicating successful transport of the synthesized NA-probe through these transporters.

Iron uptake mediated by the plant-derived chelator nicotianamine in the small intestine.

Iron is an essential metal for all living organisms that is absorbed in the intestinal cells as a heme-chelated or free form. It is unclear how important plant-derived chelators, such as nicotianamine (NA), an organic small molecule that is ubiquitous in crops, vegetables, and various other foods, contribute to iron bioavailability in mammals. We performed electrophysiological assays with Xenopus laevis oocytes and radioactive tracer experiments with Caco-2 cells. The findings revealed that the proton-coupled amino acid transporter SLC36A1 (PAT1) transports iron in the form of NA-Fe (II) complex in vitro. Decreased expression of hPAT1 by RNA interference in Caco-2 cells reduced the uptake of NA-59Fe (II) complex. The uptake of inorganic 59Fe (II) was relatively unaffected. These results imply that PAT1 transports iron as a NA-Fe (II) complex. The rate of 59Fe absorption in the spleen, liver, and kidney was higher when mice were orally administered NA-59Fe (II) compared with free 59Fe (II). The profile of site-specific PAT1 expression in the mouse intestine coincided with those of NA and iron contents, which were the highest in the proximal jejunum. Orally administered NA-59Fe (II) complex in mice was detected in the proximal jejunum by thin layer chromatography. In contrast, much less 59Fe (or NA) was detected in the duodenum, where the divalent metal transporter SLC11A2 (DMT1) absorbs free Fe (II). The collective results revealed the role of PAT1 in NA-Fe (II) absorption in the intestine and potential implication of NA in iron uptake in mammals.

Concise Total Synthesis of Tronocarpine.

A concise total synthesis of tronocarpine, a chippiine-type indole alkaloid, was accomplished. The key feature of this total synthesis is a one-pot construction of the pentacyclic skeleton containing an azabicyclo[3.3.1]nonane core by tandem cyclization from an indole derivative with all carbon side chains and functional groups. This tandem cyclization consists of α,ß-unsaturated aldehyde formation, intramolecular aldol reaction, six-membered lactamization, azide reduction, and seven-membered lactamization. The stereochemical outcome in this tandem cyclization is controlled by the stereocenter at the C14 position. This strategy can be utilized to synthesize other chippiine-type alkaloids with azabicyclo[3.3.1]nonane skeletons.

Synthesis and Antimicrobial Evaluation of Side-Chain Derivatives based on Eurotiumide A.

Side-chain derivatives of eurotiumide A, a dihydroisochroman-type natural product, have been synthesized and their antimicrobial activities described. Sixteen derivatives were synthesized from a key intermediate of the total synthesis of eurotiumide A, and their antimicrobial activities against two Gram-positive bacteria, methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), and a Gram-negative bacterium, Porphyromonas gingivalis, were evaluated. The results showed that derivatives having an iodine atom on their aromatic ring instead of the prenyl moiety displayed better antimicrobial activity than eurotiumide A against MSSA and P. gingivalis. Moreover, we discovered that a derivative with an isopentyl side chain, which is a hydrogenated product of eurotiumide A, is the strongest antimicrobial agent against all three strains, including MRSA.

Asymmetric Total Syntheses and Structure Elucidations of (+)-Eurotiumide F and (+)-Eurotiumide G.

Asymmetric total syntheses of dihydropyran containing natural products, (+)-eurotiumide F and (+)-eurotiumide G have been described. These total syntheses revealed the absolute configuration of eurotiumide F and G, and confirmed the reported structure of eurotiumide F and revised the reported structure of eurotiumide G. Highlight of these syntheses is thermal rearrangement with 4-methoxyisochroman-1-one derivative having propargyl ether on phenolic ether under thermal condition to construct dihydropyran ring. X-Ray crystallographic analysis of (+)-eurotiumide G clarified the stereochemistry at the C1-position.

Direct Synthesis of Polycyclic Tropinones by a Condensation-[4+3]-Cycloaddition Cascade Reaction.

A concise method of constructing polycyclic tropinone frameworks was developed. The single-step synthesis of polycyclic tropinone consists of an intramolecular [4+3] cycloaddition reaction of N-nosyl-pyrrole with oxyallyl cation that was generated in situ by an intermolecular condensation reaction of the nucleophilic functional groups on a tethered pyrrole with the aldehyde of 2-(silyloxy)-acrolein. This cascade reaction afforded various polycyclic tropinones including tri-, tetra-, and pentacyclic systems in high yields as single diastereomers.

Synthesis and Evaluation of a 1,3a,6a-Triazapentalene (TAP)-Bonded System.

A method of synthesizing a directly connected 1,3a,6a-triazapentalene (TAP) ring system as a linearly bonded aromatic system with a planar form was established. Various TAP-dimers and a 2-alkyl-TAP-trimer were synthesized and their fluorescence properties were evaluated. Although the direct connection of the TAP ring with other TAP rings did not affect the fluorescence properties in diluted solvent, TAP-dimers showed unique fluorescence properties derived from the aggregation state under highly concentrated conditions. In particular, TAP-dimer 5 f showed aggregation-induced emission in highly concentrated solution, and 5 b showed typical mechanochromic fluorescence in the solid state despite their compact molecular size.

Organic Chemistry Research on the Mechanistic Elucidation of Iron Acquisition in Barley.

An organic chemistry approach to the mechanistic elucidation of iron acquisition in graminaceous plants is introduced here. To elucidate this detailed mechanism using phytosiderophores, the efficient synthesis of 2'-deoxymugineic acid (DMA), a phytosiderophore of rice, was established. The synthetic DMA was confirmed to have similar iron transport activity to that of natural mugineic acid (MA). It was also revealed that the addition of synthetic DMA, along with iron, to a rice hydroponic solution enabled the rice to grow well even under an alkaline condition, and DMA clearly showed its high potential as a fertilizer to improve food production. On the other hand, the 2'-hydroxy group of MA was confirmed to serve as a point of introduction for labeling, allowing the synthesis of various mugineic acid derivatives as molecular probes. The incorporation of fluorescent mugineic acid into cells allowed them to be clearly observed by fluorescence confocal analysis, and this provided the first direct experimental evidence of transporter-mediated internalization of mugineic acid into cells.

2'-Deoxymugineic acid promotes growth of rice (Oryza sativa L.) by orchestrating iron and nitrate uptake processes under high pH conditions.

Poaceae plants release 2'-deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little information is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrate that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil-plant analysis development (SPAD) values after treatment with 3-30 µm DMA at high pH (pH 8.0), compared with untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT-PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high-affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions.

Functional 1,3a,6a-triazapentalene scaffold: Design of fluorescent probes for kinesin spindle protein (KSP).

1,3a,6a-Triazapentalene is a compact fluorescent chromophore. In this study, triazapentalene was used to modify a series of biphenyl-type inhibitors of kinesin spindle protein (KSP) to develop fluorescent probes for the intracellular visualization of this protein. Microscopic studies demonstrated that these novel triazapentalene-labeled compounds exhibited inhibitory activity towards KSP in cultured cells and provided important information concerning the intracellular distribution.

Substituent Effect at the C4-Position of 1,3a,6a-Triazapentalene.

Various 2,4-disubstituted-1,3a,6a-triazapentalenes possessing methyl and phenyl groups at the C4-position were synthesized. Fluorescence observation of the synthetic 4-methyl- and 4-phenyl-1,3a,6a-triazapentalenes revealed that the introduction of a substituent at the C4-position allowed a long-wavelength shift of the fluorescence maximum. Furthermore, the phenyl group at the C4-position was found to induce a substantial increase in the extinction coefficient value.

Synthesis of functionalized 2-vinyl-2,3-dihydropyrroles and 3-methylene-1,2,3,4-tetrahydropyridines by palladium-catalyzed cyclization of ß-enaminocarbonyl compounds with allylic bisacetates.

A palladium-catalyzed cyclization of ß-enaminocarbonyl compounds with allylic bisacetates is described. 2-Vinyl-2,3-dihydropyrroles and 3-methylene-1,2,3,4-tetrahydropyridines were produced from the reaction of ß-enaminocarbonyl compounds with 1,4-diacetoxy-2-butene and 2-methylene-1,3-propanediol diacetate, respectively.

One-pot synthesis of tri- and tetrasubstituted pyridines by sequential ring-opening/cyclization/oxidation of N-arylmethyl 3-aziridinylpropiolate esters.

A novel strategy for the one-pot synthesis of substituted pyridines from N-arylmethyl 3-aziridinylpropiolate esters is described. The method employs a three-step procedure including the formation of allenyl imines, phosphine-catalyzed cyclization, and subsequent oxidation of the dihydropyridines. Depending on the reaction conditions of the final oxidation step, tri- and tetrasubstituted pyridines can be selectively produced.

Inactivation of siderophore iron-chelating moieties by the fungal wheat root symbiont Pyrenophora biseptata.

We investigated the ability of four plant and soil-associated fungi to modify or degrade siderophore structures leading to reduced siderophore iron-affinity in iron-limited and iron-replete cultures. Pyrenophora biseptata, a melanized fungus from wheat roots, was effective in inactivating siderophore iron-chelating moieties. In the supernatant solution, the tris-hydroxamate siderophore desferrioxamine B (DFOB) underwent a stepwise reduction of the three hydroxamate groups in DFOB to amides leading to a progressive loss in iron affinity. A mechanism is suggested based on the formation of transient ferrous iron followed by reduction of the siderophore hydroxamate groups during fungal high-affinity reductive iron uptake. P. biseptata also produced its own tris-hydroxamate siderophores (neocoprogen I and II, coprogen and dimerum acid) in iron-limited media and we observed loss of hydroxamate chelating groups during incubation in a manner analogous to DFOB. A redox-based reaction was also involved with the tris-catecholate siderophore protochelin in which oxidation of the catechol groups to quinones was observed. The new siderophore inactivating activity of the wheat symbiont P. biseptata is potentially widespread among fungi with implications for the availability of iron to plants and the surrounding microbiome in siderophore-rich environments.

Synthesis of substituted tetrahydrocyclobuta[b]benzofurans by palladium-catalyzed substitution/[2+2] cycloaddition of propargylic carbonates with 2-vinylphenols.

Radical methods: The title reaction proceeds in the presence of a palladium catalyst to deliver substituted tetrahydrocyclobuta[b]benzofurans in a stereoselective manner. A radical mechanism is discussed.

1,3a,6a-Triazapentalene derivatives as photo-induced cytotoxic small fluorescent dyes.

1,3a,6a-Triazapentalene (TAP) is a compact fluorescent chromophore whose fluorescence properties vary greatly depending on the substituents on the TAP ring. This study investigated the photo-induced cytotoxicities of various TAP derivatives. Among the derivatives, 2-p-nitrophenyl-TAP showed significant cytotoxicity to HeLa cells under UV irradiation but no cytotoxicity without UV. In addition, the photo-induced cytotoxicity of 2-p-nitirophenyl-TAP was found to be cancer cell selective and effective against HeLa cells and HCT 116 cells. Under UV irradiation, 2-p-nitrophenyl-TAP generated reactive oxygen species (ROS) that induced an apoptosis and ferroptosis in cancer cells. Therefore, it was revealed that 2-p-nitrophenyl-TAP is the most compact dye that can generate ROS by photoirradiation.

Total Syntheses of Proposed Structures of 4,10-Dihydroxy-8,12-guaianolides.

The first total syntheses of two 4,10-dihydroxy-8,12-guaianolides that were reported to be natural products were achieved. Toward the syntheses of a collection of related guaianolides, the typical 5,7-fused system of 8,12-guaianolides was constructed by a ring expansion reaction of a hydroxylated coronafacic acid analogue that can be practically synthesized and optically resolved. The total syntheses of these compounds revealed that the previously reported structures of both natural products were incorrect.

Uptake mechanism of iron-phytosiderophore from the soil based on the structure of yellow stripe transporter.

Calcareous soils cover one-third of all land and cause severe growth defects in plants due to the poor water solubility of iron at high pH. Poaceae species use a unique chelation strategy, whereby plants secrete a high-affinity metal chelator, known as phytosiderophores (mugineic acids), and reabsorb the iron-phytosiderophore complex by the yellow stripe 1/yellow stripe 1-like (YS1/YSL) transporter for efficient uptake of iron from the soil. Here, we present three cryo-electron microscopy structures of barley YS1 (HvYS1) in the apo state, in complex with an iron-phytosiderophore complex, Fe(III)-deoxymugineic acid (Fe(III)-DMA), and in complex with the iron-bound synthetic DMA analog (Fe(III)-PDMA). The structures reveal a homodimeric assembly mediated through an anti-parallel ß-sheet interaction with cholesterol hemisuccinate. Each protomer adopts an outward open conformation, and Fe(III)-DMA is bound near the extracellular space in the central cavity. Fe(III)-PDMA occupies the same binding site as Fe(III)-DMA, demonstrating that PDMA can function as a potent fertilizer in an essentially identical manner to DMA. Our results provide a structural framework for iron-phytosiderophore recognition and transport by YS1/YSL transporters, which will enable the rational design of new, high-potency fertilizers.

Direct synthesis of fluorescent 1,3a,6a-triazapentalene derivatives via click-cyclization-aromatization cascade reaction.

An efficient and versatile method was established for the preparation of 1,3a,6a-triazapentalenes. The 1,3a,6a-triazapentalene skeleton without an additional fused ring system was discovered to be a compact and highly fluorescent chromophore, which exhibited various interesting fluorescent properties such as a noteworthy correlation of luminescent wavelength with the Hammett σ(p) value and a strongly positive solvatofluorochromism.