Selective Alkene Insertion into Inert Hydrogen–Metal Bonds Catalyzed by Mono(phosphorus ligand)palladium(0) Complexes

Isolated mono­(phosphorus ligand)­palladium(0) complexes catalyzed alkene insertions into hydrogen–tungsten bonds. These insertions using WHCp­(CO)₃ with ethyl acrylate and dimethyl fumarate smoothly gave the corresponding alkyltungsten complexes. Kinetic studies involving the stoichiometric reactions and DFT calculations suggest the following steps: (i) formation of a mono­(phosphorus ligand)­mono­(alkene)­palladium(0) species, (ii) subsequent reaction of a metal hydride with the palladium(0), (iii) insertion of the coordinated alkene into the resulting palladium hydride, and (iv) reductive elimination between the alkyl and metal on the palladium center to release the alkylmetal species with regeneration of a palladium(0) by a reaction with alkene

Selective Alkene Insertion into Inert Hydrogen–Metal Bonds Catalyzed by Mono(phosphorus ligand)palladium(0) Complexes

Isolated mono­(phosphorus ligand)­palladium(0) complexes catalyzed alkene insertions into hydrogen–tungsten bonds. These insertions using WHCp­(CO)₃ with ethyl acrylate and dimethyl fumarate smoothly gave the corresponding alkyltungsten complexes. Kinetic studies involving the stoichiometric reactions and DFT calculations suggest the following steps: (i) formation of a mono­(phosphorus ligand)­mono­(alkene)­palladium(0) species, (ii) subsequent reaction of a metal hydride with the palladium(0), (iii) insertion of the coordinated alkene into the resulting palladium hydride, and (iv) reductive elimination between the alkyl and metal on the palladium center to release the alkylmetal species with regeneration of a palladium(0) by a reaction with alkene

DataSheet_1_slr2103, a homolog of type-2 diacylglycerol acyltransferase genes, for plastoquinone-related neutral lipid synthesis and NaCl-stress acclimatization in a cyanobacterium, Synechocystis sp. PCC 6803.pdf

A cyanobacterium, Synechocystis sp. PCC 6803, contains a lipid with triacylglycerol-like TLC mobility but its identity and physiological roles remain unknown. Here, on ESI-positive LC-MS2 analysis, it is shown that the triacylglycerol-like lipid (lipid X) is related to plastoquinone and can be grouped into two subclasses, Xa and Xb, the latter of which is esterified by 16:0 and 18:0. This study further shows that a Synechocystis homolog of type-2 diacylglycerol acyltransferase genes, slr2103, is essential for lipid X synthesis: lipid X disappears in a Synechocystis slr2103-disruptant whereas it appears in an slr2103-overexpressing transformant (OE) of Synechococcus elongatus PCC 7942 that intrinsically lacks lipid X. The slr2103 disruption causes Synechocystis cells to accumulate plastoquinone-C at an abnormally high level whereas slr2103 overexpression in Synechococcus causes the cells to almost completely lose it. It is thus deduced that slr2103 encodes a novel acyltransferase that esterifies 16:0 or 18:0 with plastoquinone-C for the synthesis of lipid Xb. Characterization of the slr2103-disruptant in Synechocystis shows that slr2103 contributes to sedimented-cell growth in a static culture, and to bloom-like structure formation and its expansion by promoting cell aggregation and floatation upon imposition of saline stress (0.3-0.6 M NaCl). These observations provide a basis for elucidation of the molecular mechanism of a novel cyanobacterial strategy to acclimatize to saline stress, and one for development of a system of seawater-utilization and economical harvesting of cyanobacterial cells with high-value added compounds, or blooming control of toxic cyanobacteria.</p

Table_1_slr2103, a homolog of type-2 diacylglycerol acyltransferase genes, for plastoquinone-related neutral lipid synthesis and NaCl-stress acclimatization in a cyanobacterium, Synechocystis sp. PCC 6803.xlsx

A cyanobacterium, Synechocystis sp. PCC 6803, contains a lipid with triacylglycerol-like TLC mobility but its identity and physiological roles remain unknown. Here, on ESI-positive LC-MS2 analysis, it is shown that the triacylglycerol-like lipid (lipid X) is related to plastoquinone and can be grouped into two subclasses, Xa and Xb, the latter of which is esterified by 16:0 and 18:0. This study further shows that a Synechocystis homolog of type-2 diacylglycerol acyltransferase genes, slr2103, is essential for lipid X synthesis: lipid X disappears in a Synechocystis slr2103-disruptant whereas it appears in an slr2103-overexpressing transformant (OE) of Synechococcus elongatus PCC 7942 that intrinsically lacks lipid X. The slr2103 disruption causes Synechocystis cells to accumulate plastoquinone-C at an abnormally high level whereas slr2103 overexpression in Synechococcus causes the cells to almost completely lose it. It is thus deduced that slr2103 encodes a novel acyltransferase that esterifies 16:0 or 18:0 with plastoquinone-C for the synthesis of lipid Xb. Characterization of the slr2103-disruptant in Synechocystis shows that slr2103 contributes to sedimented-cell growth in a static culture, and to bloom-like structure formation and its expansion by promoting cell aggregation and floatation upon imposition of saline stress (0.3-0.6 M NaCl). These observations provide a basis for elucidation of the molecular mechanism of a novel cyanobacterial strategy to acclimatize to saline stress, and one for development of a system of seawater-utilization and economical harvesting of cyanobacterial cells with high-value added compounds, or blooming control of toxic cyanobacteria.</p

Table_2_slr2103, a homolog of type-2 diacylglycerol acyltransferase genes, for plastoquinone-related neutral lipid synthesis and NaCl-stress acclimatization in a cyanobacterium, Synechocystis sp. PCC 6803.xlsx

A cyanobacterium, Synechocystis sp. PCC 6803, contains a lipid with triacylglycerol-like TLC mobility but its identity and physiological roles remain unknown. Here, on ESI-positive LC-MS2 analysis, it is shown that the triacylglycerol-like lipid (lipid X) is related to plastoquinone and can be grouped into two subclasses, Xa and Xb, the latter of which is esterified by 16:0 and 18:0. This study further shows that a Synechocystis homolog of type-2 diacylglycerol acyltransferase genes, slr2103, is essential for lipid X synthesis: lipid X disappears in a Synechocystis slr2103-disruptant whereas it appears in an slr2103-overexpressing transformant (OE) of Synechococcus elongatus PCC 7942 that intrinsically lacks lipid X. The slr2103 disruption causes Synechocystis cells to accumulate plastoquinone-C at an abnormally high level whereas slr2103 overexpression in Synechococcus causes the cells to almost completely lose it. It is thus deduced that slr2103 encodes a novel acyltransferase that esterifies 16:0 or 18:0 with plastoquinone-C for the synthesis of lipid Xb. Characterization of the slr2103-disruptant in Synechocystis shows that slr2103 contributes to sedimented-cell growth in a static culture, and to bloom-like structure formation and its expansion by promoting cell aggregation and floatation upon imposition of saline stress (0.3-0.6 M NaCl). These observations provide a basis for elucidation of the molecular mechanism of a novel cyanobacterial strategy to acclimatize to saline stress, and one for development of a system of seawater-utilization and economical harvesting of cyanobacterial cells with high-value added compounds, or blooming control of toxic cyanobacteria.</p

A Novel Antidiabetic Drug, Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1

<div><p>Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca²⁺ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs <i>in vivo.</i> Point mutations of FFAR1 differentially affected Ca²⁺ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.</p></div

Fasiglifam does not exacerbate FFA-induced apoptotic signaling in MIN6 cells.

<p>Caspase 3/7 activity in the mouse pancreatic β cell line MIN6 after 72-h exposure to 0.25–1 mM palmitic acid (<i>A</i>) or γ-linolenic acid (γ-LA) (<i>B</i>) in combination with fasiglifam (Fas, 0.1–10 µM). “PA+Fas” and “γ-LA+Fas” indicate “1 mM palmitic acid +10 µM fasiglifam” and “1 mM γ-LA +10 µM fasiglifam”, respectively. Data shown are mean ± s.e.m. (n = 3).</p

Partial agonist activity of fasiglifam is affected by FFAR1/GPR40 expression levels.

<p>(<i>A</i>) The chemical structure of fasiglifam. (<i>B</i> and <i>C</i>) FFAR1 agonist activities of fasiglifam and free fatty acids (FFAs) in the intracellular Ca²⁺ mobilization assay using CHO cell lines expressing hFFAR1 (clone #104) (<i>B</i>) or mFFAR1 (<i>C</i>). Data are representative of three experiments. (<i>D</i>) hFFAR1 mRNA levels of hFFAR1-expressing CHO clones were evaluated by qRT-PCR. (<i>E</i><b>-</b><i>H</i>) Relative Ca²⁺ influx activities of γ-LA and fasiglifam in CHO clones #104 (<i>E</i>), #19 (<i>F</i>), #2 (<i>G</i>), and #4 (<i>H</i>) with various hFFAR1 expression levels. Error bars indicate s.e.m. (n = 3).</p

Insulinotropic effects of fasiglifam are attenuated by pharmacological reduction of plasma FFA levels <i>in vivo</i>.

<p>(<i>A</i>) Effects of the lipolysis inhibitors acipimox (30 mg/kg) and fasiglifam (10 mg/kg) on plasma free fatty acids (FFAs) during the oral glucose tolerance test (OGTT) in N-STZ-1.5 rats. (<i>B</i>) Area under the curve (AUC) of plasma FFA during 0–120 min. Fas, fasiglifam. (<i>C</i>) Plasma glucose levels after coadministration of acipimox (30 mg/kg) and fasiglifam (10 mg/kg). (<i>D</i>) AUC of plasma glucose levels during 0–120 min. *<i>P</i><0.05, **<i>P</i><0.01 versus vehicle by Student’s t-test, ^$$<i>P</i><0.01 versus vehicle by Aspin–Welch test. (<i>E</i>) Plasma insulin concentrations after coadministration of acipimox and fasiglifam during OGTT. (<i>F</i>) Insulinotropic effects of fasiglifam (Fas) just before glucose load (time 0) shown in (<i>E</i>) in the absence and presence of acipimox. **<i>P</i><0.01 versus vehicle, ^$<i>P</i><0.05 versus acipimox alone by Student’s t-test, followed by Bonferroni’s correction for four time point comparisons. Data represent mean ± s.e.m. (n = 6).</p

Point mutations of FFAR1/GPR40 differentially affect Ca²⁺ influx activities of fasiglifam and γ-LA.

<p>(<i>A</i>) Relative cell surface expression levels of FLAG-tagged FFAR1 wild<b>-</b>type and mutant receptors in transfected HEK293T cells were determined using flow cytometric analysis (FACS). (<i>B</i><b>-</b><i>J</i>) Effects of FFAR1 point mutations on the Ca²⁺ influx activities of FFAR1 agonists. HEK293T cells were transiently transfected with mock vector (<i>B</i>), wild-type (<i>C</i>), S8A (<i>D</i>), Y91A (<i>E</i>), H137A (<i>F</i>), R183A (<i>G</i>), L186F (<i>H</i>), N244A (<i>I</i>), and R258A (<i>J</i>) constructs. Data are representative of three independent experiments. Error bars indicate s.e.m. (n = 3); γ-LA, γ-linolenic acid.</p