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.

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.

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.

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.

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.

Efficient construction of the hexacyclic ring core of palau'amine: the pK a concept for proceeding with unfavorable equilibrium reactions.

Palau'amine has received a great deal of attention as an attractive synthetic target due to its intriguing molecular architecture and significant immunosuppressive activity, and we achieved its total synthesis in 2015. However, the synthesized palau'amine has not been readily applicable to the mechanistic study of immunosuppressive activity, because it requires 45 longest linear steps from a commercially available compound. Here, we report the short-step construction of the ABCDEF hexacyclic ring core of palau'amine. The construction of the CDE tricyclic ring core in a single step is achieved by our pK a concept for proceeding with unfavorable equilibrium reactions, and a palau'amine analog without the aminomethyl and chloride groups is synthesized in 20 longest linear steps from the same starting material. The palau'amine analog is confirmed to retain the immunosuppressive activity. The present synthetic approach for a palau'amine analog has the potential for use in the development of palau'amine probes for mechanistic elucidation.

Development of a mugineic acid family phytosiderophore analog as an iron fertilizer.

Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)•2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.

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.

Development of a 1,3a,6a-triazapentalene derivative as a compact and thiol-specific fluorescent labeling reagent.

For the fluorescence imaging of biologically active small compounds, the development of compact fluorophores that do not perturb bioactivity is required. Here we report a compact derivative of fluorescent 1,3a,6a-triazapentalenes, 2-isobutenylcarbonyl-1,3a,6a-triazapentalene (TAP-VK1), as a fluorescent labeling reagent. The reaction of TAP-VK1 with various aliphatic thiols proceeds smoothly to afford the corresponding 1,4-adducts in high yields, and nucleophiles other than thiols do not react. After the addition of thiol groups in dichloromethane, the emission maximum of TAP-VK1 shifts to a shorter wavelength and the fluorescence intensity is substantially increased. The utility of TAP-VK1 as a compact fluorescent labeling reagent is clearly demonstrated by the labeling of Captopril, which is a small molecular drug for hypertension. The successful imaging of Captopril, one of the most compact drugs, in this study demonstrates the usefulness of compact fluorophores for mechanistic studies.

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.

A Concise Asymmetric Total Synthesis of (+)-Epilupinine.

Asymmetric total synthesis of (+)-epilupinine was achieved in just three steps using only commercially available common reagents. The total synthesis involved alkylations of N-nosylamide, ozone oxidation, and sequential reactions of the removal of the nosyl group, intramolecular dehydrative condensation, intramolecular Mannich reaction catalyzed by l-proline, and a reduction.

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.

Quantitation of sevoflurane in whole blood and aqueous solutions by volatile organic compound sensing.

INTRODUCTION: It is difficult to quantify poorly soluble volatile anesthetics in aqueous solutions; this necessitates the development of alternative prompt methods to analyze the in vivo blood concentrations of anesthetics for the clinical assessment of anesthesia depth. In this study, we demonstrated that the difficulties can be overcome by using volatile organic compound (VOC) sensors, which allow the levels of vaporized VOCs to be quantified in several seconds and obviate the need for conventional techniques such as gas chromatography or nuclear magnetic resonance (NMR). METHODS: The concentrations of a volatile general anesthetic (sevoflurane) in aqueous solutions containing human blood components and rabbit blood were measured using a VOC sensor and those in distilled water and phosphatidylcholine suspension were compared to those determined by NMR. RESULTS: For all aqueous solutions with concentrations of up to 5 mM, the relationship between the VOC content and sevoflurane concentration was represented by a straight line passing through the origin. The concentration of sevoflurane determined by VOC sensing was well correlated with the values obtained by NMR at <1 mM, which is within the clinically relevant concentration levels. DISCUSSION: Considering the results from this study, we can conclude that VOC sensing may be useful for measuring intraoperative blood anesthetic concentrations.

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.

The helical propensity of the extracellular loop is responsible for the substrate specificity of Fe(III)-phytosiderophore transporters.

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter of Fe(III)-phytosiderophores in barley that is responsible for iron acquisition from the soil. In contrast, maize Zea mays, yellow stripe 1 (ZmYS1) possesses broad substrate specificity. In this study, a quantitative evaluation of the transport activities of HvYS1 and ZmYS1 chimera proteins revealed that the seventh extracellular membrane loop is essential for substrate specificity. The loop peptides of both transporters were prepared and analysed by circular dichroism and NMR. The spectra revealed a higher propensity for α-helical conformation of the HvYS1 loop peptide and a largely disordered structure for that of ZmYS1. These structural differences are potentially responsible for the substrate specificities of the transporters.

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.

Effective Cellular Morphology Analysis for Differentiation Processes by a Fluorescent 1,3a,6a-Triazapentalene Derivative Probe in Live Cells.

Nuclear and cytoplasmic morphological changes provide important information about cell differentiation processes, cell functions, and signal responses. There is a strong desire to develop a rapid and simple method for visualizing cytoplasmic and nuclear morphology. Here, we developed a novel and rapid method for probing cellular morphological changes of live cell differentiation process by a fluorescent probe, TAP-4PH, a 1,3a,6a-triazapentalene derivative. TAP-4PH showed high fluorescence in cytoplasmic area, and visualized cytoplasmic and nuclear morphological changes of live cells during differentiation. We demonstrated that TAP-4PH visualized dendritic axon and spine formation in neuronal differentiation, and nuclear structural changes during neutrophilic differentiation. We also showed that the utility of TAP-4PH for visualization of cytoplasmic and nuclear morphologies of various type of live cells. Our visualizing method has no toxicity and no influence on the cellular differentiation and function. The cell morphology can be rapidly observed after addition of TAP-4PH and can continue to be observed in the presence of TAP-4PH in cell culture medium. Moreover, TAP-4PH can be easily removed after observation by washing for subsequent biological assay. Taken together, these results demonstrate that our visualization method is a powerful tool to probe differentiation processes before subsequent biological assay in live cells.