Facile synthesis of diiodoheteroindenes and understanding their Sonogashira cross-coupling selectivity for the construction of unsymmetrical enediynes.

Electrophile-promoted cyclizations of functionalized alkynes offer a useful tool for constructing halogen-substituted heterocycles primed for further derivatization. Preinstallation of an iodo-substituent at the alkyne prior to iodo-cyclization opens access to ortho di-iodinated heterocyclic precursors for the preparation of unsymmetrical heterocycle-fused enediynes. This general approach was used to prepare 2,3-diiodobenzothiophene, 2,3-diiodoindole, and 2,3-diiodobenzofuran, a useful family of substrates for systematic studies of the role of heteroatoms on the regioselectivity of cross-coupling reactions. Diiodobenzothiophene showed much higher regioselectivity for Sonogashira cross-coupling at C2 than diiodoindole and diiodobenzofuran. As a result, benzothiophene can be conveniently involved in a one-pot sequential coupling with two different alkynes, yielding unsymmetrical benzothiophene-fused enediynes. On the other hand, the Sonogashira reaction of diiodoindole and diiodobenzofuran formed considerable amounts of di-substituted enediynes in addition to the monoalkyne product by coupling at C2. Interestingly, no C3-monocoupling products were observed for all of the diiodides, suggesting that the incorporation of the 1st alkyne at C2 activates the C3 position for the 2nd coupling. Additional factors affecting regioselectivity were detected, discussed and connected, through computational analysis, to transmetalation being the rate-determining step for the Sonogashira reaction. Several enediynes synthesized showed cytotoxic activity, which is not associated with DNA strand breaks typical of natural enediyne antibiotics.

Effect of Biotin Starvation on Gene Expression in Komagataella phaffii Cells.

Methylotrophic yeast Komagataella phaffii is widely used in biotechnology for recombinant protein production. Due to the practical significance of these yeasts, it is extremely important to properly select cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on gene expression in K. phaffii at transcriptome level was investigated. It was demonstrated, that the response of K. phaffii cell to biotin deficiency strongly depends on the carbon source in the medium. In the media containing glycerol, biotin deficiency led to activation of the genes involved in biotin metabolism, glyoxylate cycle, and synthesis of acetyl-CoA in cytoplasm, as well as repression of the genes, involved in lipo- and gluconeogenesis. In the methanol-containing media, biotin deficiency primarily led to repression of the genes, involved in protein synthesis, and activation of cell response to oxidative stress.

4,5-Bis(arylethynyl)-1,2,3-triazoles-A New Class of Fluorescent Labels: Synthesis and Applications.

Cu-catalyzed 1,3-dipolar cycloaddition of ethyl 2-azidoacetate to iodobuta-1,3-diynes and subsequent Sonogashira cross-coupling were used to synthesize a large series of new triazole-based push-pull chromophores: 4,5-bis(arylethynyl)-1H-1,2,3-triazoles. The study of their optical properties revealed that all molecules have fluorescence properties, the Stokes shift values of which exceed 150 nm. The fluorescent properties of triazoles are easily adjustable depending on the nature of the substituents attached to aryl rings of the arylethynyl moieties at the C4 and C5 atoms of the triazole core. The possibility of 4,5-bis(arylethynyl)-1,2,3-triazoles' application for labeling was demonstrated using proteins and the HEK293 cell line. The results of an MTT test on two distinct cell lines, HEK293 and HeLa, revealed the low cytotoxicity of 4,5-bis(arylethynyl)triazoles, which makes them promising fluorescent tags for labeling and tracking biomolecules.

4-Azidocinnoline-Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe.

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide-amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Design and Synthesis of New 5-aryl-4-Arylethynyl-1H-1,2,3-triazoles with Valuable Photophysical and Biological Properties.

Cu-catalyzed 1,3-dipolar cycloaddition of methyl 2-azidoacetate to iodobuta-1,3-diynes and subsequent Suzuki-Miyaura cross-coupling were used to synthesize new triazoles derivatives: 5-aryl-4-arylethynyl-1H-1,2,3-triazoles. Investigation of their optical properties by using UV absorption and fluorescence emission spectroscopies revealed that all molecules possess fluorescence properties with the values of the Stokes shift more than 100 nm. The photophysical behavior of the two most promising triazoles in polar and non-polar solvents was also studied.

Cytokinin biosynthesis genes expressed during nodule organogenesis are directly regulated by the KNOX3 protein in Medicago truncatula.

Cytokinin is an important regulator of symbiotic nodule development. Recently, KNOTTED1-LIKE HOMEOBOX 3 transcription factor (TF) was shown to regulate symbiotic nodule development possibly via the activation of cytokinin biosynthesis genes. However, the direct interaction between the KNOX3 TF and its target genes has not been investigated up to date. Here, using EMSA analysis and SPR-based assay, we found that MtKNOX3 homeodomain directly binds to the regulatory sequences of the MtLOG1, MtLOG2, and MtIPT3 genes involved in nodulation in Medicago truncatula. Moreover, we showed that MtLOG2 and MtIPT3 expression patterns partially overlap with MtKNOX3 expression in developing nodules as it was shown by promoter:GUS analysis. Our data suggest that MtKNOX3 TF may directly activate the MtLOG1, MtLOG2, and MtIPT3 genes during nodulation thereby increasing cytokinin biosynthesis in developing nodules.

Synthesis and Properties of 6-Aryl-4-azidocinnolines and 6-Aryl-4-(1,2,3-1H-triazol-1-yl)cinnolines.

An efficient approach towards the synthesis of 6-aryl-4-azidocinnolines was developed with the aim of exploring the photophysical properties of 6-aryl-4-azidocinnolines and their click reaction products with alkynes, 6-aryl-4-(1,2,3-1H-triazol-1-yl)cinnolines. The synthetic route is based on the Richter-type cyclization of 2-ethynyl-4-aryltriazenes with the formation of 4-bromo-6-arylcinnolines and nucleophilic substitution of a bromine atom with an azide functional group. The developed synthetic approach is tolerant to variations of functional groups on the aryl moiety. The resulting azidocinnolines were found to be reactive in both CuAAC with terminal alkynes and SPAAC with diazacyclononyne, yielding 4-triazolylcinnolines. It was found that 4-azido-6-arylcinnolines possess weak fluorescent properties, while conversion of the azido function into a triazole ring led to complete fluorescence quenching. The lack of fluorescence in triazoles could be explained by the non-planar structure of triazolylcinnolines and a possible photoinduced electron transfer (PET) mechanism. Among the series of 4-triazolylcinnoline derivatives a compound bearing hydroxyalkyl substituent at triazole ring was found to be cytotoxic to HeLa cells.

Relative Reactivity of Benzothiophene-Fused Enediynes in the Bergman Cyclization.

To find promising analogues of naturally occurring enediyne antibiotics with a sufficient reactivity in the Bergman cyclization and moderately stable under isolation and storage, a scale of relative enediynes reactivity was created on the basis of calculated free activation energies for the Bergman cyclization within 12 known and new benozothiophene, benzene, and cinnoline annulated 9- and 10-membered enediynes. To verify the predicted reactivity/stability balance, three new carbocyclic enediynes fused to a benzothiophene core bearing 3,4,5-trimethoxybenzene, fluoroisopropyl, and isopropenyl substituents were synthesized using the Nicholas-type macrocyclization. It was confirmed that annulation of a 3,4,5-trimethoxybenzene moiety to a 10-membered enediyne macrocycle imparts high reactivity to an enediyne while also conferring instability under ambient temperature. Fluoroisopropyl-substituted 10-membered enediyne from the opposite end of the scale was found to be stable while moderately reactive in the Bergman cyclization. Along with the experimentally confirmed moderate reactivity (DSC kinetic studies), (fluoroisopropyl)enediyne showed a significant DNA damaging activity in plasmid cleavage assays comparable with the known anticancer drug Zeocin.

Crystal structure of (1S,4S)-2,5-diazo-niabi-cyclo[2.2.1]heptane dibromide.

The cage of 2,5-di-aza-bicyclo-[2.2.1]heptane is frequently employed in synthetic chemistry as a rigid bicyclic counterpart of the piperazine ring. The 2,5-di-azabicyclo-[2.2.1]heptane scaffold is incorporated into a variety of compounds having pharmacological and catalytic applications. The unsubstituted parent ring of the system, 2,5-di-aza-bicyclo-[2.2.1]heptane itself, has not been structurally characterized. We herein report on the mol-ecular structure of the parent ring in (1S,4S)-2,5-diazo-niabi-cyclo-[2.2.1]heptane dibromide, C5H12N22+·2Br-. The asymmetric unit contains two crystallographically independent cages of 2,5-di-aza-bicyclo-[2.2.1]heptane. Each cage is protonated at the two nitro-gen sites. The overall charge balance is maintained by four crystallographically independent bromide ions. In the crystal, the components of the structure are linked via a complex three-dimensional network of N-H⋯Br hydrogen bonds.

Crystal structure of (1R,5S)-endo-(8-methyl-8-azoniabi-cyclo-[3.2.1]oct-3-yl)ammonium aqua-tri-chlorido-nitratocopper(II).

The structure of a salt of diprotonated endo-3-amino-tropane crystallized with a copper(II) anionic cluster is reported, viz. (C8H18N2)[CuCl3(NO3)(H2O)]. Neither ion in the salt has been structurally characterized previously. In the crystal, the ions pack together to form a three-dimensional structure held together by a network of inter-molecular N-H⋯O, O-H⋯Cl and N-H⋯Cl hydrogen-bonding inter-actions. Selective crystallization of the title compound can be considered as a simple method for the separation of the exo and endo isomers of 3-amino-tropane.

The 7-aza-norbornane nucleus of epibatidine: 7-aza-bicyclo-[2.2.1]heptan-7-ium chloride.

7-Aza-bicyclo-[2.2.1]heptane (7-aza-norbornane) is a bridged heterocyclic nucleus found in epibatidine, the alkaloid isolated from the skin of the tropical poison frog Epipedobates tricolor. Since epibatidine is known as one of the most potent acetyl-choline nicotinic receptor agonists, a plethora of literature has been devoted to this alkaloid. However, there are no structural data on the unsubstituted 7-aza-norbornane, the parent bicyclic ring of epibatidine and its derivatives. We herein present the structural characterization of the 7-aza-bicyclo-[2.2.1]heptane parent ring as its hydro-chloride salt, namely 7-aza-bicyclo-[2.2.1]heptan-7-ium chloride, C6H12N+·Cl-. The compete cation is generated by a crystallographic mirror plane with the N atom lying on the mirror, as does the chloride anion. In the crystal, the cations are linked to the anions by N-H⋯Cl hydrogen bonds, which generate [001] chains.

Between Adamantane and Atrane: Intrabridgehead Interactions in the Cage-Like Phosphane Related to a Novel Tris(homoadamantane) Ring System.

Herein, we report unusual four-center interactions in the novel cage-like phosphane, 1,4,7-triaza-9-phosphatricyclo[5.3.2.1(4,9) ]tridecane (CAP). This water-soluble ligand, the first example of a tris(homoadamantane) ring system, can be considered a macrocyclic homologue of the well-known PTA (1,3,5-triaza-7-phosphaadamantane). However, (31) P NMR spectroscopic anomalies of CAP follow those typical for the bi-/tricyclic atrane systems. Another atrane-like feature of CAP is the ability of one nitrogen atom to undergo out-in pyramidal inversion. The latter is associated with a substantial decrease in the intracage N-N and P-N distances. Analysis of electron density distribution [molecular electrostatic potential (MESP) and atoms-in-molecules (AIM) approaches] suggests that the P and N atoms in the pyramidally inverted CAP derivatives are involved in interactions resulting in accumulation of electron density at the center of the phosphane cage. The latter can reliably explain the stereoelectronic and NMR anomalies of the new ligand. The semi-flexible CAP cage populates the structural niche between the rigid adamantine skeleton of PTA and flexible atrane systems and can be regarded as an alternative to PTA in aqueous coordination chemistry.