Iridium-Catalyzed [2 + 2 + 2] Cycloaddition of Bithiophen-Linked Diynes with Nitriles: Scope and Mechanistic Study with Quantum Chemical Calculation.

We report a simple and atom-efficient method for the synthesis of bithiophene-fused isoquinolines by iridium-catalyzed [2 + 2 + 2] cycloaddition of bithiophene-linked diynes with nitriles. All three structural isomers of bithiophene-linked diynes underwent [2 + 2 + 2] cycloaddition, and the trend in the reactivity for cycloaddition was diyne 1 = diyne 3 > diyne 2. Dibenzothiophene-linked diyne also reacted with nitriles to form a variety of cycloadducts. Cycloaddition of bithiophene-linked diynes with alkynes and an isocyanate formed naphthodithiophenes and a 2-pyridone derivative, respectively. Cycloadducts bearing a 2-aminopyridine moiety and benzothiophene rings showed intense fluorescence at around 530 nm and gave a fluorescence quantum yield of 0.44. Furthermore, quantum chemical calculations provided insight into the origin of the difference in reactivity of three bithiophene-linked diynes. The different reactivities of the three diynes 1-3 are believed to originate from the step where an iridacyclopentadiene reacts with a coordinated nitrile to form azairidabicyclo[3.2.0]heptatriene. HOMOs of iridacyclopentadiene play a decisive role in this step.

High Phase-Purity and Composition-Tunable Ferromagnetic Icosahedral Quasicrystal.

We discovered a ferromagnetic Au-Ga-Dy icosahedral quasicrystal (i QC), not only with high phase purity but also with tunable composition. The isothermal magnetization of the polycrystalline ferromagnetic i QC was closely investigated and the mean-field-like nature of the ferromagnetic transition is elucidated. Moreover, the maximum Weiss temperature (θ_{p}) of the i QCs was found at the electrons-per-atom (e/a) ratio of 1.70 being well consistent with those of ACs, validating tunability of the magnetic properties of i QCs on the basis of θ_{p}-e/a scheme for the first time. Thus, the present work provided direct evidence that the magnetism of the i QCs depends on the e/a ratio or the Fermi energy, paving the way for future studies on various exotic magnetic textures formed on a quasiperiodic lattice through the e/a ratio.

Iridium-Catalyzed Branch-Selective Hydroalkylation of Simple Alkenes with Malonic Amides and Malonic Esters.

We report the iridium-catalyzed branch-selective hydroalkylation of simple alkenes such as aliphatic alkenes and aromatic alkenes with malonic amides and malonic esters under neutral reaction conditions. A variety of aliphatic alkenes and aromatic alkenes bearing bromine, chlorine, ester, 2-thienylcarboxylate, silyl, and phthalimide groups were all found to be suitable for this hydroalkylation. The combination of this method with Krapcho dealkoxycarbonylation realized a one-pot synthesis of ß-substituted amide and ester from ß-amide ester and malonic ester. The hydroalkylated products derived from malonic amides are suitable for further transformation. The finely tuned reaction conditions realized the selective transformation of hydroalkylated products to 1,3-diamines or monoamides with the same reagent. Deuterium labeling experiments and measurement of the kinetic isotope effect indicated that the catalytic cycle involves a reversible step and cleavage of the C-H bond is not a rate-determining step. Density functional theory calculations provided insight into the reaction mechanism, where the carboiridation step is followed by C-H reductive elimination.

Synthesis of azafluoranthenes by iridium-catalyzed [2 + 2 + 2] cycloaddition and evaluation of their fluorescence properties.

We report a method for the synthesis of azafluoranthenes under neutral reaction conditions in a highly atom-economical manner by the iridium-catalyzed [2 + 2 + 2] cycloaddition of 1,8-dialkynylnaphthalenes with nitriles. A variety of nitriles react with methyl- or phenyl-substituted 1,8-dialkynylnaphthalenes to give a wide range of azafluoranthenes. Azafluoranthenes bearing an amino group show intense fluorescence at around 500 nm. Comparison of the fluorescence properties of amine-substituted azafluoranthenes with related compounds revealed the importance of the amine moiety for obtaining a high fluorescence quantum yield. The choice of the solvent affected the emission maxima and the fluorescence quantum yield. Azafluoranthenes bearing pyrrolidine exhibited blue-shifted emission bands in a non-polar solvent and gave a fluorescence quantum yield of 0.76 in toluene. A Lippert-Mataga plot and computational studies provide insight into the origin of the fluorescence of azafluoranthenes. Furthermore, cellular experiments using human breast adenocarcinoma cells SK-BR-3 demonstrated the feasibility of using azafluoranthenes as fluorescent probes.

Effects of Two Electron-Donating and/or -Withdrawing Substituents on Two-Photon Absorption for Diphenylacetylene Derivatives.

Two-photon absorption for diphenylacetylene (DPA) derivatives with two substituents (-OMe and/or -NO2) at the 4,4'-position was investigated experimentally and theoretically. The two-photon absorption spectra and the two-photon absorption cross-sections σ(2) for DPA derivatives were obtained by optical-probing photoacoustic spectroscopy (OPPAS). The simulated two-photon absorption spectra of the DPA derivatives, obtained with the time-dependent density functional theory within the Tamm-Dancoff approximation, agreed well with the experimental ones. The mechanisms for enhancement of the σ(2) for centrosymmetric and non-centrosymmetric DPA derivatives were found to be different. The large σ(2) for centrosymmetric molecules (DPA-OMeOMe and DPA-NO2NO2) results from the magnitude of the transition dipole moment, while for non-centrosymmetric molecules (DPA-OMeNO2), it is enhanced by the smaller detuning energy. Information on two-photon absorption properties of DPA derivatives obtained in this study will be important for the molecular design of two-photon absorption materials.

A Substituent Effect on Two-Photon Absorption of Diphenylacetylene Derivatives with an Electron-Donating/Withdrawing Group.

Two-photon absorption for diphenylacetylene derivatives with an electron-donating (ED) or electron-withdrawing (EW) group (DPA-Rs) was investigated by high-sensitivity optical-probing photoacoustic spectroscopy. Two-photon absorption spectra and two-photon absorption cross sections σ(2) for DPA-Rs were successfully obtained. Two-photon absorption spectra of DPA-Rs with stronger ED or EW groups display more significant red-shifts and larger σ(2) values. Simulated two-photon absorption spectra, using time-dependent density functional theory within the Tamm-Dancoff approximation, compared well with the experimental spectra. Based on the three-state model, the substituent effect on the two-photon absorption for DPA-Rs was expected to manifest in the transition dipole moments and detuning energies. Information obtained from investigating the monosubstituent effect on two-photon absorption of DPA is critical for an improved understanding of two-photon absorption.

Biocontainment of genetically modified organisms by synthetic protein design.

Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

Two-photon absorption property of Cl-substituted diphenylacetylenes by optical-probing photoacoustic spectroscopy.

Two-photon absorption spectra and two-photon absorption cross sections of Cl-substituted diphenylacetylenes (ClDPAs) were investigated by optical-probing photoacoustic spectroscopy and quantum chemical calculations for the first time. The two-photon absorption spectra of ClDPAs exhibited intense two-photon absorption bands at around 480 nm, which are forbidden by one-photon absorption. The two-photon absorption cross sections σ(2) of o-, m-, and p-ClDPAs at 476 nm were determined to be 22 ± 1, 23 ± 1, and 38 ± 2 GM, respectively. Compared with diphenylacetylene (DPA) (27 GM at 472 nm), the σ(2) values of o- and m-ClDPAs were lower, while that of p-ClDPA was higher. Simulated two-photon absorption spectra using the TD-B3LYP/6-311+G(d,p) level of calculations within the Tamm-Dancoff approximation, based on the three-state model, well agreed with the experimental results. The difference in the σ(2) values of DPA and ClDPAs was responsible for those in the transition dipole moments between the intermediate and the final states.

Synthesis of Chiral Homoallylic Nitriles by Iridium-Catalyzed Allylation of Cyanoacetates.

A synthesis of chiral homoallylic nitriles by the iridium-catalyzed allylation of cyanoacetates followed by Krapcho demethoxycarbonylation has been developed. A wide range of homoallylic nitriles were obtained with a high enantioselectivity (>95-99% ee). These compounds are useful chiral building blocks because further synthetic elaboration starting from a nitrile or terminal alkene is possible.

Synthesis of Multisubstituted Azatriphenylenes by Iridium-Catalyzed [2 + 2 + 2] Cycloaddition of Biaryl-Linked Diynes with Nitriles.

A convenient synthesis of multisubstituted azatriphenylenes is reported. [Ir(cod)Cl]2/diphosphine is an efficient catalyst for the [2 + 2 + 2] cycloaddition of biaryl-linked diynes with nitriles to give multisubstituted azatriphenylenes in high yields. Aromatic, heteroaromatic, aliphatic, and functionalized nitriles could be used for the reaction.

Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization.

LAGLIDADG homing endonucleases (meganucleases) are sequence-specific DNA cleavage enzymes used for genome engineering. Recently, meganucleases fused to transcription activator-like effectors have been demonstrated to efficiently introduce targeted genome modifications. However, retargeting meganucleases to genomic sequences of interest remains challenging because it usually requires extensive alteration of a large number of amino acid residues that are situated in and near the DNA interface. Here we describe an effective strategy to extensively redesign such an extensive biomolecular interface. Well-characterized meganucleases are computationally screened to identify the best candidate enzyme to target a genomic region; that protein is then redesigned using iterative rounds of in vitro selections within compartmentalized aqueous droplets, which enable screening of extremely large numbers of protein variants at each step. The utility of this approach is illustrated by engineering three different meganucleases to cleave three human genomic sites (found in two exons and one flanking intron in two clinically relevant genes) and a fourth endonuclease that discriminates between single-nucleotide polymorphism variants of one of those targets. Fusion with transcription activator-like effector DNA binding domains significantly enhances targeted modification induced by meganucleases engineered in this study. Simultaneous expression of two such fusion endonucleases results in efficient excision of a defined genomic region.

Iridium-Catalyzed Synthesis of Acylpyridines by [2 + 2 + 2] Cycloaddition of Diynes with Acyl Cyanides.

2-Acylpyridines were prepared by iridium-catalyzed [2 + 2 + 2] cycloaddition of α,ω-diynes with acyl cyanides. [Ir(cod)Cl]2/rac-BINAP or F-DPPE is an efficient catalyst for this reaction. The scope and limitations of this reaction have been disclosed.

Exploitation of binding energy for catalysis and design.

Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme-DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (K(D)) and the Michaelis constant under single-turnover conditions (K(M)*), and those in the (+) half-site primarily decrease the turnover number (k(cat)*). The reduction of activity produced by mutations on the (-) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the use of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (-) side do so by modulating K(M)*, whereas redesigns with altered specificities on the (+) side modulate k(cat)*. Our results illustrate how classical enzymology and modern protein design can each inform the other.

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.

Tapping natural reservoirs of homing endonucleases for targeted gene modification.

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for "genome editing" in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

(8-Benzoyl-2,7-dimeth-oxy-naphthalen-1-yl)(4-phen-oxy-phen-yl)methanone.

In the mol-ecule of the title compound, C32H24O5, the benzoyl group and the 4-phenoxy substituted benzoyl group at the 1- and 8-positions of the naphthalene ring system are aligned almost anti-parallel. The two benzene rings make a dihedral angle of 21.18 (10)°, and are inclined to the naphthalene ring system by 86.53 (9) and 82.95 (8)°, respectively. In the crystal, C-H⋯O inter-actions are observed involving aromatic and meth-oxy H atoms with ketonic carbonyl O atoms, as well as C-H⋯π inter-actions between aromatic H atoms and the π-systems of naphthalene and benzene rings. These interactions form a three-dimensional architecture and afford a waved alignment of the naphthalene ring systems along the c axis.

{2,7-Dimeth-oxy-8-[4-(propan-2-yl-oxy)benzo-yl]naphthalen-1-yl}[4-(propan-2-yl-oxy)phen-yl]methanone.

The title compound, C32H32O6, crystallized with two independent molecules in the asymmetric unit. Each molecule has essentially the same feature of non-coplanar aromatic rings whereby the two 4-isopropoxybenzoyl groups are twisted in a perpendicular manner to the naphthalene ring and oriented in the same direction (syn-orientation). The benzene rings of the aroyl groups make dihedral angles of 16.13 (7) and 25.31 (7)° in the two molecules. These benzene rings make dihedral angles of 88.38 (8) and 75.32 (7)° with the naphthalene ring system in one molecule, and 89.71 (7) and 82.11 (7)° in the other. In the crystal, mol-ecules are linked via C-H⋯O hydrogen bonds, forming a three-dimensional network. In one independent molecule, the 2-propyl groups of both isoprop-oxy groups are disordered over two positions with site occupancies of 0.512 (3) and 0.488 (3).

{2,7-Dieth-oxy-8-[(naphthalen-1-yl)carbon-yl]naph-thalen-1-yl}(naphthalen-1-yl)methanone.

In the title compound, C36H28O4, the 1-naphthoyl groups at the 1- and 8-positions of the central 2,7-dieth-oxy-naphthalene ring system are aligned almost anti-parallel and make a dihedral angle of 76.59 (4)°. The dihedral angles between the central 2,7-dieth-oxy-naphthalene ring system and the terminal naphthalene ring systems are 86.48 (4) and 83.97 (4)°. In the crystal, C-H⋯π inter-actions between the central naphthalene ring systems and the naphthoyl groups are observed along the a axis, with the mol-ecules forming a columnar structure. The columns are linked into chains parallel to the b axis by C-H⋯O inter-actions.

Preemptively planned en bloc resection of an extensive right adrenal pheochromocytoma involving the right hepatic division, caval thrombus and segmental caudal vena cava in a dog with Budd-Chiari-like syndrome.

BACKGROUND: Surgical resection is the treatment of choice for canine adrenal pheochromocytomas (PHEOs). Information on en bloc resection of adrenal PHEO with tumour thrombus, right hepatic division and segmental caudal vena cava (CVC) running through the adrenal tumour and right hepatic division is limited. OBJECTIVE: To describe the preemptively planned en bloc resection of an extensive right adrenal PHEO involving the right hepatic division, the caval thrombus and the segmental CVC in a dog with Budd-Chiari-like syndrome (BCLS). METHODS: A 13-year-old castrated male miniature dachshund was referred for surgical treatment due to anorexia, lethargy and severe abdominal distension caused by abundant ascites. Preoperative computed tomography (CT) revealed a large mass in the right adrenal gland with a large caval thrombus obstructing the CVC and hepatic veins, which caused BCLS. Additionally, collateral vessels were formed between the CVC and azygos veins. No findings suggested obvious metastases. Based on CT findings, an en bloc resection of the adrenal tumour with caval thrombus, right hepatic division and segmental CVC was planned. RESULTS: The preoperatively planned resection was feasible; the tumour was completely resected grossly. The operation time and total Pringle manoeuvre time were 162 min and 16 min 56 s, respectively. There was no postoperative hindlimb oedema, renal dysfunction, ascites or abdominal distention. The patient's clinical signs, including appetite, fully improved. Hospitalization lasted 16 days. However, the patient died on the 130th postoperative day due to suspected metastases and cachexia. CONCLUSIONS: Even in case of an extensive infiltration of adrenal PHEO causing BCLS, an en bloc resection might be successfully achieved based on the preoperative CT findings speculating the collateral vessels formed for caudal venous return.