Efficient Synthesis and Structural Analysis of Chiral 4,4'-Biazulene.

4,4'-Biazulene is a potentially attractive key component of an axially chiral biaryl compound, however, its structure and properties have not been clarified owing to the lack of its efficient synthesis. We report a breakthrough in the reliable synthesis of 4,4'-biazulene, which is achieved by the access to azulen-4-ylboronic acid pinacol ester and 4-iodoazulene as novel key synthetic intermediates for the Suzuki-Miyaura cross-coupling reaction. The X-ray crystallographic analysis of 4,4'-biazulene confirmed its axial chirality. The enantiomers of 4,4'-biazulene were successfully resolved by HPLC on the chiral stationary phase column. The kinetic experiments and DFT calculations indicate that the racemization energy barrier of 4,4'-biazulene is comparable to that of 1,1'-binaphthyl.

Invariant natural killer T cells stimulated with cholesteryl glycosides modulate immune responses in allergy and delayed-type hypersensitivity.

Invariant NKT cells were stimulated with cholesteryl O-acyl α-glycosides in the context of CD1d. The activated NKT cells have potential to sustain the homeostasis in the body exposed to excess in either Th1- or Th2-immunity.

Synthesis of 2-Iodoazulenes by the Iododeboronation of Azulen-2-ylboronic Acid Pinacol Esters with Copper(I) Iodide.

Azulen-2-ylboronic acid pinacol ester, prepared by iridium-catalyzed C-H borylation of azulene, efficiently underwent iododeboronation with a stoichiometric amount of copper(I) iodide. This reaction allowed the synthesis of 2-iodoazulene in only two steps starting from azulene. This methodology was successfully applied to analogous azulenes.

Synthesis of Alkylated Pyrimidines via Photoinduced Coupling Using Benzophenone as a Mediator.

The synthesis of alkylated pyrimidines was achieved via benzophenone-mediated photoinduced coupling between saturated heterocycles and sulfonylpyrimidines. The pyrimidine ring was selectively introduced at the nonacidic C(sp3)-H bond proximal to heteroatoms including oxygen, nitrogen, and sulfur. This is a coupling reaction mediated solely by photoexcited benzophenone, an organic molecule, without the aid of any metallic catalysts or reagents.

Photo-induced Substitutive Introduction of the Aldoxime Functional Group to Carbon Chains: A Formal Formylation of Non-Acidic C(sp(3) )-H Bonds.

A photo-induced substitutive introduction of an aldoxime functional group to carbon chains was achieved using photo-excited 4-benzoylpyridine as a C(sp(3) )-H bond cleaving agent and arylsulfonyl oxime as an aldoxime precursor. The non-acidic C-H bonds in various substances, including cycloalkanes, ethers, azacycles, and cyclic sulfides, were chemoselectively converted at ambient temperature under neutral conditions. The present transformation is a formal formylation of non-acidic C(sp(3) )-H bonds in a single step.

Photochemically induced radical alkynylation of C(sp3)-H bonds.

A general strategy for photochemical alkynylation of unreactive C(sp(3))-H bonds has been developed. After C-H abstraction by the photo-excited benzophenone, a two-carbon unit was efficiently transferred to the generated radical from 1-tosyl-2-(trimethylsilyl)acetylene to afford the alkynylated product. The present reaction enables construction of various tri- and tetra-substituted carbons from heteroatom-substituted methylenes, methines and alkanes in a highly chemoselective fashion, and would serve as a new synthetic strategy for rapid construction of complex structures.

Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans.

Male embryonic mice with mutations in the X-linked aristaless-related homeobox gene (Arx) developed with small brains due to suppressed proliferation and regional deficiencies in the forebrain. These mice also showed aberrant migration and differentiation of interneurons containing gamma-aminobutyric acid (GABAergic interneurons) in the ganglionic eminence and neocortex as well as abnormal testicular differentiation. These characteristics recapitulate some of the clinical features of X-linked lissencephaly with abnormal genitalia (XLAG) in humans. We found multiple loss-of-function mutations in ARX in individuals affected with XLAG and in some female relatives, and conclude that mutation of ARX causes XLAG. The present report is, to our knowledge, the first to use phenotypic analysis of a knockout mouse to identify a gene associated with an X-linked human brain malformation.

C-type lectin Mincle-dependent and -independent activation of invariant NKT cells by exposure to Helicobacter pylori α-cholesteryl glucosides.

Helicobacter pylori extracts cholesterol from the host and converts it to its glycosides. We found that cholesteryl 6'-O-acyl α-glucoside (ChAcαG) produced by H. pylori is recognised by both invariant Vα14+ NKT (iNKT) cells and a C-type lectin receptor Mincle (Clec4e). However, it is unclear how these duplicated recognitions cooperate and contribute to host defence against H. pylori. Among T cell populations in the liver, iNKT cells predominantly expressed the T cell activation marker CD69 just after stimulation with ChAcαG. The production of IFN-γ and IL-4 was strictly dependent on both CD1d and Jα18 expressions, indicating the necessity of iNKT cell activation for the initiation of immune responses. Production of IFN-γ by iNKT cells was markedly reduced by the Mincle deficiency on antigen-presenting cells (APCs), while IL-4 production was not significantly influenced. IL-2 production by iNKT cell hybridomas was also diminished by the Mincle deficiency upon stimulation with APCs previously loaded with ChAcαG. Here, the immune responses of iNKT cell hybridomas stimulated with wild-type APCs were reduced by the addition of anti-IL-12 blocking antibody to the level stimulated with Mincle-deficient APCs. Collectively, these results suggest that iNKT cells can be activated with the cholesteryl glycosides via a Mincle-dependent, IL-12 signal-dependent pathway and a Mincle-independent, invariant TCR signal-dominant pathway. iNKT cells activated via the Mincle-dependent pathway produce IFN-γ-dominant cytokines; hence, they may contribute to enhancing proinflammatory responses against H. pylori infection.

Radical amination of C(sp3)-H bonds using N-hydroxyphthalimide and dialkyl azodicarboxylate.

A direct conversion of C(sp(3))-H bonds to C(sp(3))-N bonds has been achieved by utilizing catalytic N-hydroxyphthalimide (NHPI) and stoichiometric dialkyl azodicarboxylate. NHPI functions as a precursor of the electron-deficient phthalimide N-oxyl radical (PINO) to abstract hydrogens, and dialkyl azodicarboxylate acts as a trapping agent of the resultant carbon radical to generate the hydrazine derivatives. This C-H amination proceeds in a highly chemoselective manner with a wide applicability to functionalize benzylic, propargylic, and aliphatic C-H bonds. Furthermore, the obtained hydrazine compounds were readily converted to the corresponding carbamates or amines. Hence, the present protocol for direct introduction of the nitrogen functionality serves as a powerful tool for efficient construction of nitrogen-substituted natural products and pharmaceuticals.

Ring opening of cyclic vinylogous acyl triflates using stabilized carbanion nucleophiles: claisen condensation linked to carbon-carbon bond cleavage.

Addition of stabilized carbanionic nucleophiles to cyclic vinylogous acyl triflates (VATs) triggers a ring-opening fragmentation to give acyclic beta-keto ester and related products, much like those observed traditionally in the Claisen condensation. Unlike in the classical Claisen condensation, however, the VAT-Claisen reaction described herein is rendered irreversible by C-C bond cleavage, not by deprotonation of the activated methylene product. Full details of this original reaction methodology are disclosed herein, including how subtle differences between the various nucleophiles impact the proper choice of reaction conditions for making 1,3-diketones, beta-keto esters, and beta-keto phosphonates.

Fertility of spermatozoa cryopreserved with 2% acetamide or glycerol through artificial insemination in the Japanese white rabbit.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P < 0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P < 0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.

RNAi-mediated knockdown of mouse melanocortin-4 receptor in vitro and in vivo, using an siRNA expression construct based on the mir-187 precursor.

RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken ß-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells. Using this construct, we generated a transgenic mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions. Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore, the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that this siRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies in vitro as well as in vivo.

Claisen-type condensation of vinylogous acyl triflates.

[reaction: see text] The Claisen-type condensation reaction of cyclic vinylogous carboxylic acid triflates with lithium enolates and their analogues produces acyclic alkynes bearing a 1,3-diketone-type moiety. The present transformation is proposed to proceed via a 1,2-addition of the enolate to the vinylogous acyl triflate, followed by fragmentation of the aldolate intermediate.

Aryl Ketone Catalyzed Radical Allylation of C(sp3)-H Bonds under Photoirradiation.

The catalytic introduction of an allyl group at nonacidic C(sp3)-H bonds was achieved under photoirradiation, in which 1,2-bis(phenylsulfonyl)-2-propene acts as an allyl source and 5,7,12,14-pentacenetetrone (PT) works as a C-H bond-cleaving catalyst. A variety of substances, including alkanes, carbamates, ethers, sulfides, and alcohols, were chemoselectively allylated in a single step under neutral conditions. The present transformation is catalyzed solely by an organic molecule, PT, and proceeds smoothly even under visible light irradiation (425 nm) in the case of alkanes as a starting substance.

Alkylation of Nonacidic C(sp3)-H Bonds by Photoinduced Catalytic Michael-Type Radical Addition.

Photoinduced catalytic Michael-type radical addition was achieved via olefin insertion into a nonacidic C(sp3)-H bond, utilizing 2-chloroanthraquinone as a C-H bond-cleaving catalyst and 1,1-bis(phenylsulfonyl)ethylene as an olefinic substrate. The present radical protocol allows carbon chain extension stemming from nonacidic C-H bonds, which complements alkylation at acidic C-H bonds under ionic conditions and installs the active methine site that acts as a versatile synthetic handle for further transformations.

Photoinduced Oxidation of Secondary Alcohols Using 4-Benzoylpyridine as an Oxidant.

Photoinduced oxidation of secondary alcohols to ketones was achieved by utilizing an equimolar amount of 4-benzoylpyridine as an oxidant. This transformation proceeds at ambient temperature and exhibits high compatibility with polar functionalities including benzoyl, silyl, and methoxymethyl alcohol protecting groups as well as tosyloxy, bromo, sulfonyl, carbamate, ester, and carboxylic acid units. The present oxidation is solely promoted by the action of organic molecules without the aid of metallic reagents.

Photochemically induced radical transformation of C(sp3)-H bonds to C(sp3)-CN bonds.

A general protocol for direct transformation of unreactive C(sp(3))-H bonds to C(sp(3))-CN bonds has been developed. The C-H activation was effected by photoexcited benzophenone, and the generated carbon radical was subsequently trapped with tosyl cyanide to afford the corresponding nitrile in a highly efficient manner. The present methodology is widely applicable to versatile starting materials and, thus, serves as a powerful tool for selective one-carbon elongation for construction of architecturally complex molecules.

CCl3CN: a crucial promoter of mCPBA-mediated direct ether oxidation.

The direct oxidation of ether sp(3) C-H bonds using the new reagent system mCPBA/CCl(3)CN/MeCN has been developed. CCl(3)CN in MeCN drastically alters the reactivity of m-chloroperbenzoic acid (mCPBA), and chemoselective transformation of methyl ethers to ketones was realized under mild conditions. Radical-based mCPBA-mediated oxidation was suggested as the reaction mechanism. The present new reaction expands the utility of methyl ethers as stable synthetic precursors of carbonyl compounds and of mCPBA as a radical-based C-H oxidizing agent.