Cyclization Reactions of Coordinated Alkynes in Tungsten(II) Complexes

Displacement of a CO ligand from [Tp‘(CO)2W{HC⋮CH}][OTf] with iodide leads to the neutral parent acetylene complex Tp‘(CO)(I)W{HC⋮CH} (2). Deprotonation of 2 followed by methylation is regioselective and yields a single isomer of the propyne complex Tp‘(CO)(I)W{HC⋮CCH3} (3). Deprotonation of 3 followed by alkylation with RI (R = Me, I(CH2)n (n = 3−7)) is also regioselective and leads to a single isomer, Tp‘(CO)(I)W{RC⋮CCH3} (4−9). Deprotonation of Tp‘(CO)(I)W{I(CH2)nC⋮CCH3} (n = 5 (7), 7 (9)) leads to an η2-allenyl intermediate which undergoes intramolecular alkylation (i.e. endocyclic cyclization) to form Tp‘(CO)(I)W{cyclooctyne} (10) and Tp‘(CO)(I)W{cyclodecyne} (11), respectively. The exocyclic cyclization precursor Tp‘(CO)(I)W{PhC⋮C(CH2)5I} (12) was obtained by deprotonation of the propargyl carbon of Tp‘(CO)(I)W{PhC⋮CCH3} followed by alkylation with I(CH2)4I. The cyclopentyl derivative Tp‘(CO)(I)W{PhC⋮C(cyclopentyl)} was generated by deprotonation of 12 followed by intramolecular alkylation (i.e. exocyclic cyclization). A coordinated cyclodecyne ligand is observed in the X-ray structure of Tp‘(CO)(I)W{cyclodecyne} (11)

Regioselective and Stereoselective Reactions of 2-Butyne Bound to a Resolved Chiral Tungsten(II) Center

Deprotonation of chiral complexes of the type Tp‘(CO)(I)W{RC⋮CCH2R‘} at the propargyl carbon followed by alkylation is regioselective and stereoselective. Photolysis liberates the elaborated alkynes from the metal complex. Resolution of metal diastereomers, with amido ligands, Tp‘(CO)(NHR)W{CH3C⋮CCH3} (NH2R = ((S)-(−)-α-methylbenzylamine), has been accomplished by fractional crystallization. Conversion to an amine complex followed by iodide replacement of H2NCHMePh produces separate enantiomers of Tp‘(CO)(I)W{CH3C⋮CCH3}. Methylation followed by benzylation of each enantiomer affords Tp‘(CO)(I)W{CH3C⋮CCHMeCH2Ph}. The alkynes (−)-CH3C⋮CCHMeCH2Ph and (+)-CH3C⋮CCHMeCH2Ph have been released from each of these enantiomerically enriched complexes in optically active form, as assayed by optical rotation and by 1H NMR with chiral shift reagents. The barrier to alkyne rotation in Tp‘(CO)(I)W{RC⋮CCH2R‘} complexes has been probed by variable-temperature 1H NMR, and extended Huckel molecular orbital calculations have been performed on model complexes. The origin of regioselectivity and stereoselectivity is considered in light of the X-ray structures of Tp‘(CO)(I)W{CH3C⋮CCHMe(CH2)4I} (7) and Tp‘(CO)(I)W{Me(PhCH2)HCC⋮CCHMeCH(OH)Ph} (11). The absolute configuration of Tp‘(CO)W{CH3C⋮CCH3}(NHCHMePh) ((+)-14(SS)) has been established by X-ray analysis with the resolving amine as reference

Octahedral Ru(II) Amido Complexes TpRu(L)(L‘)(NHR) (Tp = Hydridotris(pyrazolyl)borate; L = L‘ = P(OMe)₃ or PMe₃ or L = CO and L‘ = PPh₃; R = H, Ph, or ^tBu): Synthesis, Characterization, and Reactions with Weakly Acidic C−H Bonds

The octahedral Ru(II) amine complexes [TpRu(L)(L‘)(NH2R)][OTf] (L = L‘ = PMe3, P(OMe)3 or L = CO and L‘ = PPh3; R = H or tBu) have been synthesized and characterized. Deprotonation of the amine complexes [TpRu(L)(L‘)(NH3)][OTf] or [TpRu(PMe3)2(NH2tBu)][OTf] yields the Ru(II) amido complexes TpRu(L)(L‘)(NH2) and TpRu(PMe3)2(NHtBu). Reactions of the parent amido complexes or TpRu(PMe3)2(NHtBu) with phenylacetylene at room temperature result in immediate deprotonation to form ruthenium−amine/phenylacetylide ion pairs, and heating a benzene solution of the [TpRu(PMe3)2(NH2tBu)][PhC2] ion pair results in the formation of the Ru(II) phenylacetylide complex TpRu(PMe3)2(C⋮CPh) in >90% yield. The observation that [TpRu(PMe3)2(NH2tBu)][PhC2] converts to the Ru(II) acetylide with good yield while heating the ion pairs [TpRu(L)(L‘)(NH3)][PhC2] yields multiple products is attributed to reluctant dissociation of ammonia compared with the tbutylamine ligand (i.e., different rates for acetylide/amine exchange). These results are consistent with ligand exchange reactions of Ru(II) amine complexes [TpRu(PMe3)2(NH2R)][OTf] (R = H or tBu) with acetonitrile. The previously reported phenyl amido complexes TpRuL2(NHPh) {L = PMe3 or P(OMe)3} react with 10 equiv of phenylacetylene at elevated temperature to produce Ru(II) acetylide complexes TpRuL2(C⋮CPh) in quantitative yields. Kinetic studies indicate that the reaction of TpRu(PMe3)2(NHPh) with phenylacetylene occurs via a pathway that involves TpRu(PMe3)2(OTf) or [TpRu(PMe3)2(NH2Ph)][OTf] as catalyst. Reactions of 1,4-cyclohexadiene with the Ru(II) amido complexes TpRu(L)(L‘)(NH2) (L = L‘ = PMe3 or L = CO and L‘ = PPh3) or TpRu(PMe3)2(NHtBu) at elevated temperatures result in the formation of benzene and Ru hydride complexes. TpRu(PMe3)2(H), [Tp(PMe3)2RuCC(H)Ph][OTf], [Tp(PMe3)2RuC(CH2Ph){N(H)Ph}][OTf], and [TpRu(PMe3)3][OTf] have been independently prepared and characterized. Results from solid-state X-ray diffraction studies of the complexes [TpRu(CO)(PPh3)(NH3)][OTf], [TpRu(PMe3)2(NH3)][OTf], and TpRu(CO)(PPh3)(C⋮CPh) are reported

Octahedral Ru(II) Amido Complexes TpRu(L)(L‘)(NHR) (Tp = Hydridotris(pyrazolyl)borate; L = L‘ = P(OMe)₃ or PMe₃ or L = CO and L‘ = PPh₃; R = H, Ph, or ^tBu): Synthesis, Characterization, and Reactions with Weakly Acidic C−H Bonds

The octahedral Ru(II) amine complexes [TpRu(L)(L‘)(NH2R)][OTf] (L = L‘ = PMe3, P(OMe)3 or L = CO and L‘ = PPh3; R = H or tBu) have been synthesized and characterized. Deprotonation of the amine complexes [TpRu(L)(L‘)(NH3)][OTf] or [TpRu(PMe3)2(NH2tBu)][OTf] yields the Ru(II) amido complexes TpRu(L)(L‘)(NH2) and TpRu(PMe3)2(NHtBu). Reactions of the parent amido complexes or TpRu(PMe3)2(NHtBu) with phenylacetylene at room temperature result in immediate deprotonation to form ruthenium−amine/phenylacetylide ion pairs, and heating a benzene solution of the [TpRu(PMe3)2(NH2tBu)][PhC2] ion pair results in the formation of the Ru(II) phenylacetylide complex TpRu(PMe3)2(C⋮CPh) in >90% yield. The observation that [TpRu(PMe3)2(NH2tBu)][PhC2] converts to the Ru(II) acetylide with good yield while heating the ion pairs [TpRu(L)(L‘)(NH3)][PhC2] yields multiple products is attributed to reluctant dissociation of ammonia compared with the tbutylamine ligand (i.e., different rates for acetylide/amine exchange). These results are consistent with ligand exchange reactions of Ru(II) amine complexes [TpRu(PMe3)2(NH2R)][OTf] (R = H or tBu) with acetonitrile. The previously reported phenyl amido complexes TpRuL2(NHPh) {L = PMe3 or P(OMe)3} react with 10 equiv of phenylacetylene at elevated temperature to produce Ru(II) acetylide complexes TpRuL2(C⋮CPh) in quantitative yields. Kinetic studies indicate that the reaction of TpRu(PMe3)2(NHPh) with phenylacetylene occurs via a pathway that involves TpRu(PMe3)2(OTf) or [TpRu(PMe3)2(NH2Ph)][OTf] as catalyst. Reactions of 1,4-cyclohexadiene with the Ru(II) amido complexes TpRu(L)(L‘)(NH2) (L = L‘ = PMe3 or L = CO and L‘ = PPh3) or TpRu(PMe3)2(NHtBu) at elevated temperatures result in the formation of benzene and Ru hydride complexes. TpRu(PMe3)2(H), [Tp(PMe3)2RuCC(H)Ph][OTf], [Tp(PMe3)2RuC(CH2Ph){N(H)Ph}][OTf], and [TpRu(PMe3)3][OTf] have been independently prepared and characterized. Results from solid-state X-ray diffraction studies of the complexes [TpRu(CO)(PPh3)(NH3)][OTf], [TpRu(PMe3)2(NH3)][OTf], and TpRu(CO)(PPh3)(C⋮CPh) are reported

Octahedral Ru(II) Amido Complexes TpRu(L)(L‘)(NHR) (Tp = Hydridotris(pyrazolyl)borate; L = L‘ = P(OMe)₃ or PMe₃ or L = CO and L‘ = PPh₃; R = H, Ph, or ^tBu): Synthesis, Characterization, and Reactions with Weakly Acidic C−H Bonds

The octahedral Ru(II) amine complexes [TpRu(L)(L‘)(NH2R)][OTf] (L = L‘ = PMe3, P(OMe)3 or L = CO and L‘ = PPh3; R = H or tBu) have been synthesized and characterized. Deprotonation of the amine complexes [TpRu(L)(L‘)(NH3)][OTf] or [TpRu(PMe3)2(NH2tBu)][OTf] yields the Ru(II) amido complexes TpRu(L)(L‘)(NH2) and TpRu(PMe3)2(NHtBu). Reactions of the parent amido complexes or TpRu(PMe3)2(NHtBu) with phenylacetylene at room temperature result in immediate deprotonation to form ruthenium−amine/phenylacetylide ion pairs, and heating a benzene solution of the [TpRu(PMe3)2(NH2tBu)][PhC2] ion pair results in the formation of the Ru(II) phenylacetylide complex TpRu(PMe3)2(C⋮CPh) in >90% yield. The observation that [TpRu(PMe3)2(NH2tBu)][PhC2] converts to the Ru(II) acetylide with good yield while heating the ion pairs [TpRu(L)(L‘)(NH3)][PhC2] yields multiple products is attributed to reluctant dissociation of ammonia compared with the tbutylamine ligand (i.e., different rates for acetylide/amine exchange). These results are consistent with ligand exchange reactions of Ru(II) amine complexes [TpRu(PMe3)2(NH2R)][OTf] (R = H or tBu) with acetonitrile. The previously reported phenyl amido complexes TpRuL2(NHPh) {L = PMe3 or P(OMe)3} react with 10 equiv of phenylacetylene at elevated temperature to produce Ru(II) acetylide complexes TpRuL2(C⋮CPh) in quantitative yields. Kinetic studies indicate that the reaction of TpRu(PMe3)2(NHPh) with phenylacetylene occurs via a pathway that involves TpRu(PMe3)2(OTf) or [TpRu(PMe3)2(NH2Ph)][OTf] as catalyst. Reactions of 1,4-cyclohexadiene with the Ru(II) amido complexes TpRu(L)(L‘)(NH2) (L = L‘ = PMe3 or L = CO and L‘ = PPh3) or TpRu(PMe3)2(NHtBu) at elevated temperatures result in the formation of benzene and Ru hydride complexes. TpRu(PMe3)2(H), [Tp(PMe3)2RuCC(H)Ph][OTf], [Tp(PMe3)2RuC(CH2Ph){N(H)Ph}][OTf], and [TpRu(PMe3)3][OTf] have been independently prepared and characterized. Results from solid-state X-ray diffraction studies of the complexes [TpRu(CO)(PPh3)(NH3)][OTf], [TpRu(PMe3)2(NH3)][OTf], and TpRu(CO)(PPh3)(C⋮CPh) are reported

Regulation of midgut cell proliferation impacts <i>Aedes aegypti</i> susceptibility to dengue virus

<div><p><i>Aedes aegypti</i> is the vector of some of the most important vector-borne diseases like dengue, chikungunya, zika and yellow fever, affecting millions of people worldwide. The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission. We studied the homeostatic response of the midgut against oxidative stress, as well as bacterial and dengue virus (DENV) infections, focusing on the proliferative ability of the intestinal stem cells (ISC). Inhibition of the peritrophic matrix (PM) formation led to an increase in reactive oxygen species (ROS) production by the epithelial cells in response to contact with the resident microbiota, suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance. We show that dengue virus infection induces midgut cell division in both DENV susceptible (Rockefeller) and refractory (Orlando) mosquito strains. However, the susceptible strain delays the activation of the regeneration process compared with the refractory strain. Impairment of the Delta/Notch signaling, by silencing the Notch ligand Delta using RNAi, significantly increased the susceptibility of the refractory strains to DENV infection of the midgut. We propose that this cell replenishment is essential to control viral infection in the mosquito. Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges, including virus infection. In addition, we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence.</p></div

The peritrophic matrix shapes intestinal homeostasis by limiting contact of the gut epithelium with the microbiota and preventing ROS production.

Red strain mosquitoes were fed on normal blood or blood infected with non-pathogenic S. marcescens or entomopathogenic P. entomophila bacteria. Another group of mosquitoes was fed blood supplemented with heat-killed P. entomophila. The midguts were dissected 24 hours after feeding and immunostained for PH3. (A) Representative images of PH3-labeled mitotic cells (green) of the midgut epithelium 24 h after a naïve blood meal or blood infected with P. entomophila. The nuclei are stained with DAPI (blue). The arrowheads indicate PH3+ cells. Scale bar = 100 μm (B) Total PH3-positive cells were quantified from the midguts of mosquitoes fed on naïve and bacteria-infected blood (n = 25) or heat-inactivated P. entomophila. (n = 12). The medians of three independent experiments are shown. The asterisks indicate significantly different values *** P(C) Inhibition of PM formation results in a significant increase of progenitors cells under mitosis. The mosquitoes were fed blood or blood supplemented with diflubenzuron (DFB), DFB plus an antibiotic cocktail (AB) or DFB plus 50 mM ascorbate (ASC). The midguts were dissected 24 hours after feeding, and the mitotic indices were quantified by counting PH3+ cells. The medians of at least three independent experiments are shown (n = 30). The asterisks indicate significantly different values *** P(D) Assessments of reactive oxygen species in the midguts were conducted by incubating midguts of insects fed as in (C) with a 50 μM concentration of the oxidant-sensitive fluorophore DHE. (E) Quantitative analysis of the fluorescence images shown in (D) were performed using ImageJ 1.45s software (n = 7–9 insects).</p

Interference in gut homeostatic response impacts vector competence.

<p><b>(A).</b> The midguts of dsRNA-injected Rockefeller and Orlando mosquitoes were dissected 24 days after a blood meal for silencing quantification of Delta, the ligand of Notch. Total PH3-positive cells were quantified from midguts of silenced Delta or control (GFP) mosquitoes from the Rockefeller <b>(B)</b> or Orlando <b>(C)</b> strains, both 1 and 5 days after blood meal. <b>(D)</b> dsRNA-Injected mosquitoes were fed DENV2-infected blood, and 5 days after the infection, the midguts were dissected for the plaque assay. <b>(E)</b> The susceptible (Rockefeller) mosquitoes were pre-treated with the tissue-damaging dextran sulfate sodium (DSS) accordingly to material and methods section. Twelve hours after the end of the DSS treatment, the mosquitoes were fed with DENV-2-infected blood. After 5 days, the midguts were dissected for the plaque assay. <b>(F)</b> The percentage of infected midguts (infection prevalence) was scored from the same set of data as in (E). The medians of at least three independent experiments are shown. n = 20–25 in (A), (B) and (C); n = 20–26 in (D) and n = 40 in (E). Statistical analyzes used were: Student’s t-test for (A), (B) and (C); Mann-Whitney U-tests were used for infection intensity (D and E); and chi-square tests were performed to determine the significance of infection prevalence analysis (F). *P<0.05, ** P<0.01, **** P<0.0001.</p

General structure of the midgut epithelium of <i>Aedes aegypti</i> and modulation of cell proliferation upon blood meal.

<p>The midgut epithelia from a blood-fed <i>A</i>. <i>aegypti</i> females were fixed in PFA and in <b>(A)</b> sections of 0.14 μm were stained with WGA-FITC (green), red phalloidin (red) and DAPI (blue). The peritrophic matrix (PM), intestinal lumen (Lumen), polyploid enterocytes (EC) and basally localized–putative proliferative cells (*)–are visible. In <b>(B)</b>, confocal image (z-stack of 0.7 μm slides (20X)) of the two monolayers of the midgut of a blood-fed female, 5 days post feeding, stained with PH3 mouse antibody (green), DAPI (blue), and phalloidin (red)–Inset (2x): polyploid enterocytes (EC) are PH3-positive ISC (ISC) are visible. <b>(C)</b> Mosquitoes were fed on a sugar solution (10% sucrose), blood or blood supplemented with 100μM of the pro-oxidant paraquat. The insect midguts were dissected 24 hours after feeding and immunostained for PH3. Representative images of mitotic (PH3-labeled) cells (red) in the epithelial midgut of animals fed on sugar, blood or blood supplemented with paraquat are shown. The nuclei are stained with DAPI (blue). The arrowheads indicate PH3+ cells. <b>(D)</b> Quantification of PH3-positive cells per midgut of sugar, blood or blood plus paraquat-fed mosquitoes for sugar and blood and 18 for blood-paraquat fed midguts. The experiments were performed on Red Eye mosquito strain. The medians of at least three independent experiments are shown (n = 40 for sugar and blood and n = 18 for paraquat supplemented blood). The asterisks indicate significantly different values, **** P<0.0001 (Student’s t-test).</p

Dengue virus infection impacts midgut homeostasis in a strain specific manner.

<p><b>(A)</b> Blood feeding induces different levels of PH3 positive cells in the midgut of the susceptible (Rock) and refractory (Orl) strains 24 hours after the meal. Representative images of PH3 labeling in both strains 24 hours after the blood meal. The nuclei are stained with DAPI. The arrowheads indicate PH3+ cells. Scale bar = 100 μm. <b>(B)</b> Mosquitoes from the two strains were blood fed and at day zero (non blood-fed) or at different days after feeding, the midguts were dissected and immunostained for PH3. The red arrows indicate the time of blood feeding and the time in which the digestion is completed (after blood bolus excretion). In <b>(C)</b> the mosquitoes were fed on DENV2-infected blood and mitotic-cell counting was performed at different days after infection. The red arrow indicates the time of DENV escape from the midgut to hemocoel. The medians of at least three independent experiments are shown (n = 30). The asterisks indicate significantly different values * P<0.05 ** P<0.01 and *** P<0.001 (Student’s t-test).</p