Complete Molecule Research Articles

Dimethyl 4,4′-(diazenediyl)dibenzoate at 100 K

In the asymmetric part of the unit cell of the title compound, C16H14N2O4, there are two chemically equivalent but crystallographic independent half mol­ecules. The geometric centre of each complete mol­ecule lies on a crystallographic inversion centre. Both mol­ecules are almost planar [mean deviations of atoms in the two molecules are 0.032 (2) and 0.044 (2) Å] and their geometries are similar. In the crystal, mol­ecules are arranged in columns along the a axis. There are no inter­molecular donor–acceptor distances shorter than 3.4 Å.

RAMPAGE: Promoter Activity Profiling by Paired‐End Sequencing of 5′‐Complete cDNAs

RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) is a method that harnesses highly specific sequencing of 5'-complete complementary DNAs to identify transcription start sites (TSSs) genome-wide. Although TSS mapping has historically relied on detection of 5'-complete cDNAs, current genome-wide approaches typically have limited specificity and provide only scarce information regarding transcript structure. RAMPAGE allows for highly stringent selection of 5'-complete molecules, thus allowing base-resolution TSS identification with a high signal-to-noise ratio. Paired-end sequencing of medium-length cDNAs yields transcript structure information that is essential to interpreting the relationship of TSSs to annotated genes and transcripts. As opposed to standard RNA-seq, RAMPAGE explicitly yields accurate and highly reproducible expression level estimates for individual promoters. Moreover, this approach offers a streamlined 2- to 3-day protocol that is optimized for extensive sample multiplexing, and is therefore adapted for large-scale projects. This method has been applied successfully to human and Drosophila samples, and in principle should be applicable to any eukaryotic system.

A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA-damage repair and DNA replication take place on single-stranded DNA (ssDNA) or require specific single-stranded/double-stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single-molecule studies of DNA-protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single-molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force-induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force-induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA.

5,15-Bis(4-pentyloxyphenyl)porphyrin

In the title compound, C42H42N4O2, the complete molecule is generated by a crystallographic inversion centre. The porphyrin system exhibits a near planar macrocycle conformation with an average deviation from the least-squares plane of the 24 macrocycle atoms of 0.037 (5) Å. The phenyl ipso C atoms are positioned above and below the porphyrin plane by 0.35 (1) Å and the macrocycle shows evidence of in-plane rectangular elongation with N⋯N separations of 3.032 (5) and 2.803 (5) Å. Two intramolecular N—H⋯N hydrogen bonds occur.

Crystal structure of trans-diiodidobis(4-phenylpyridine-κN)platinum(II), C22H18I2N2Pt

Abstract C22H18I2N2Pt, monoclinic, P21/c (no. 14), a = 9.072(2) Å, b = 9.158(2) Å, c = 13.169(2) Å, β = 107.281(3)°, V = 1044.8 Å3, Z = 2, Rgt(F) = 0.0319, wRref(F2) = 0.0727, T = 200 K.

New Class of Molecular Conductance Switches Based on the [1,3]-Silyl Migration from Silanes to Silenes.

On the basis of first-principles density functional theory calculations, we propose a new molecular photoswitch which exploits a photochemical [1,3]-silyl(germyl) shift leading from a silane to a silene (a Si=C double bonded compound). The silanes investigated herein act as the OFF state, with tetrahedral saturated silicon atoms disrupting the conjugation through the molecules. The silenes, on the other hand, have conjugated paths spanning over the complete molecules and thus act as the ON state. We calculate ON/OFF conductance ratios in the range of 10–50 at a voltage of +1 V. In the low bias regime, the ON/OFF ratio increases to a range of 200–1150. The reverse reaction could be triggered thermally or photolytically, with the silene being slightly higher in relative energy than the silane. The calculated activation barriers for the thermal back-rearrangement of the migrating group can be tuned and are in the range 108–171 kJ/mol for the switches examined herein. The first-principles calculations together with a simple one-level model show that the high ON/OFF ratio in the molecule assembled in a solid state device is due to changes in the energy position of the frontier molecular orbitals compared to the Fermi energy of the electrodes, in combination with an increased effective coupling between the molecule and the electrodes for the ON state.

1,3-Bis[(3-chloropyrazin-2-yl)oxy]benzene

The asymmetric unit of the title compound, C14H8Cl2N4O2, contains one half-mol­ecule, the complete mol­ecule being generated by the operation of a twofold rotation axis. The Cl atom deviates significantly from the plane of the pyrazine ring [0.0215 (4) Å]. The central benzene ring makes a dihedral angle of 72.82 (7)° with the plane of the pyrazine ring.

The complete mitochondrial genome of Channa argus, Channa maculata and hybrid snakehead fish [Channa maculata (♀) × Channa argus (♂)

We sequenced and characterized the complete mitochondrial genome of Channa argus, Channa maculata and their hybrid [C. maculata (♀) and C. argus (♂)]. All the three mitochondrial genomes contained the typical complement of 13 protein-coding genes, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and 1 control region. The entire mitochondrial DNA (mtDNA) molecule of C. maculata was 16,559 bp long while the complete mtDNA molecule of C. argus and hybrid snakehead fish was 16,558 bp long. This is the first report on the complete mitogenome sequence of C. maculata and hybrid snakehead fish.

A systematic theoretical study of the electronic structures of porphyrin dimers: DFT and TD-DFT calculations on diporphyrins linked by ethane, ethene, ethyne, imine, and azo bridges

Theoretical calculations of the geometries, electronic structures and electronic absorption spectra of a series of covalently-linked porphyrin dimers are reported. The diporphyrins comprise 5,10,15-triphenylporphyrinatozinc(II) (ZnTriPP) units linked through the meso carbons by two-atom bridges, namely 1,2-ethanediyl (1), trans-1,2-ethenediyl (2), ethynediyl (3), 1,2-iminomethenediyl (4), and transdiazenediyl (5). The structures were optimised in toluene solvent by Density Functional Theory (DFT), using the integral equation formalism variant of the polarizable continuum model. The calculations were performed using the B3LYP functional and the 6-31G(d,p) basis set. The complete molecules were modelled, with no substitution of smaller groups on the periphery. In parallel, the compounds 2–5 were prepared by known or novel synthetic routes, to enable comparisons of experimental electronic absorption spectra with those calculated using time dependent-DFT at the same level of theory. As the ethane dimer 1 is not yet synthetically accessible, the model monomer meso-2-phenylethylZnTriPP was used for comparisons with the theoretical predictions. The results form a self-consistent set, enabling for the first time legitimate comparisons of the electronic structures of the series, especially regarding the degree to which the porphyrin p-systems interact by conjugation across the bridges. The theoretical calculations of the electronic transitions match the observed spectra in toluene to a remarkable degree, especially with respect to the peak maximum of the Q band, which represents to a large degree the energy of the HOMO–LUMO transition. The imine 4 is intrinsically polar due to the asymmetric bridge, and the HOMO is located almost exclusively on the ZnTriPP unit attached to the nitrogen of the imine, and the LUMO on the C-attached ring. Thus the Q-band transition is mapped as a comprehensive charge-transfer from the former ring to the latter. This may have consequences for the non-linear optical properties of the system. The azoporphyrin 5 exhibits the largest splittings between the interacting MOs via the conjugated bridge, vindicating a prediction by Anderson and co-workers in 2002, and confirmed experimentally by our synthesis of 5. The collected results also indicate that this level of theory is more thanadequate as a model with which to handle these large delocalised molecules.

Detecting low-level flexibility using residual dipolar couplings: a study of the conformation of cellobiose

We have developed novel NMR methods for the measurement of heteronuclear residual dipolar couplings (RDCs) in molecules with severely overlapping NMR resonances. These and other methods enabled us to obtain 31 RDCs for α-D-cellobiose and 24 RDCs for β-D-cellobiose. The interpretation of the data in the approximation of a rigid disaccharide structure, using RDCs and interglycosidic (3)J coupling constants, yielded conformation that is very close to that determined using X-ray crystallography. However, depending on which ring was used to calculate the order parameters, the dihedral angle ψH varied up to 30° or 40°, while the φH angle was always the same. This indicates residual flexibility of the glycosidic linkage between the two monosaccharide rings and was observed for both α- and β-D-cellobiose. The RDC analysis using rigid fragments rather than a complete molecule has thus shown that the glycosidic bond of cellobiose is not completely rigid and exhibits low-level flexibility. The sources of this flexibility are discussed and evidence presented to support a hypothesis that it is associated with the ψ more than the φ angle.

Abstract B69: Novel MHC class I antibody fusions for cancer treatment.

Abstract MHC (major histocompatibility complex) class I-restricted CD8 cytotoxic T cells recognize tumor cells or virus infected cells as foreign and consequently initiate a cascade of events resulting in their destruction. An immunotherapeutic strategy based on antibody-mediated targeting of virus-derived peptide-MHC class I complexes to tumor cells aiming at the subsequent elimination of these tumor cells by peptide-specific cytotoxic T cells has been proposed a few years ago. A crucial limitation hampering advances of this therapeutic concept was the recombinant expression of MHC class I fused full IgG immunoglobulins. We have identified novel recombinant fusion formats that allow the expression of full-length peptide-MHC class I IgG fusion molecules. These formats contain a single recombinant human pMHC class I complex consisting of a viral peptide (CMV or EBV derived), beta-2-microglobulin and HLA heavy chain (A*0201, lacking the transmembrane domain) fused to one of the two immunoglobulin heavy chains of a complete antibody molecule. These fusions can be expressed at high levels in standard mammalian expression systems overcoming several former technical hurdles (e.g. prokaryotic expression, low refolding yield, chemical coupling of components). By flow cytometry we show that the new fusion proteins successfully deliver virus-peptide complexed MHC class I molecules to tumor cells. The fusion proteins are potent CD8 T cell recruiters for human donor derived specific T cells or human PBMCs from chronically infected donors. A low frequency of virus peptide specific CD8 T cells in PBMCs (0.25 to 3.8 % of all CD8 positive T cells) effectively triggers in a peptide specific manner the killing of tumor cells at sub-nanomolar concentrations (cytotoxic T cell assay using real time analysis with xCelligence confirmed by classical LDH release). No unspecific activation of T cells was observed even at high concentrations, indicating a favorable safety profile. Confocal time-lapse microscopy showed T cell synapse formation on the tumor cells and serial killing by the T cells. We experimentally compare the killing of tumor cells of the MHC class I fused antibodies with bispecific anti-CD3 mediated T cell recruiters and characterized the virus specific T cells from human donors in detail. Our results show for the first time that recombinant pMHC class I antibody fusions in our novel full human IgG format can be expressed with a significant yield in common mammalian production cell lines. The redirection of MHC class I mediated recruitment of pre-existing virus specific CD8 T cells from human PBMCs is potent mechanism to attack tumor cells with a low risk of unspecific T cell activation making the concept now suitable as a therapeutic option. Citation Format: Martina Schmittnaegel, Eike Hoffmann, Olaf Mundigl, Gerhard Niederfellner, Klaus Bosslet, Pablo Umana, Victor Levitsky, Christian Klein, Hendrik Knoetgen. Novel MHC class I antibody fusions for cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B69.

Mitochondrial DNA- a Tool for Phylogenetic and Biodiversity Search in Equines

It is imperative to assess the maternal lineage in order to achieve a broad picture of evolution, phylogenetic and genetic biodiversity within and among different breeds of livestock. In recent past, there has been a considerable advancement in sequencing of complete mammalian mtDNA molecules and their analysis. Most of the studies have focused on the mitochondrial D-loop region, the most variable part of mtDNA due to increased substitution rate than in the rest of the mtDNA genome which serves as a better genetic marker to assess the diversity. Mitochondrial DNA (mtDNA) possesses several favorable characteristics, including large quantity in the cell, small genome size, haploid, maternal inheritance with extremely low probability of paternal leakage, higher mutation rate than nuclear DNA, and amenable to change mainly through mutation rather than recombination. All these features make mtDNA a useful and one of the most frequently used markers in molecular systematic and has been widely employed to address questions of genetic diversity, population structure and population evolution of animals including equines. Many native breeds of horses as well as ponies were assessed for their genetic diversity and ancestry on the basis of studies on mitochondrial DNA to address the questions of evolution along with breed development and conservation.

Packing principles for urea and thiourea solvates: structures of urea : morpholine (1 : 1), urea : 1,4-dioxane (1 : 1), thiourea : morpholine (4 : 3) and thiourea : 1,4-dioxane (4 : 1)

The solvents 1,4-dioxane and morpholine have been employed to synthesize solvates of urea and thiourea. The structures confirm the tendency of urea to form more rigid systems of hydrogen bonds in the plane of the N2CO moiety, thus forming layer structures with close complementarity of the donors and acceptors, whereas the more flexible sulfur acceptor of thiourea can also accept hydrogen bonds from donors that lie far from the N2CS plane, forming three-dimensional packing patterns with much more variable parameters. A database investigation confirms these tendencies. The solvate urea:morpholine (1:1) crystallizes in Pbcm with Z = 4. The complete urea molecule lies in the mirror plane, as do the heteroatoms of the morpholine molecule. The molecular packing is a layer structure. The solvate urea:1,4-dioxane (1:1) crystallizes in P2/c with Z = 2. The CO bond of the urea molecule lies along a twofold axis, whereas the dioxane molecule lies across an inversion centre. The molecules form a layer structure analogous to that of the morpholine solvate. The thiourea solvates are more complex, and both involve a more irregular hydrogen bonding geometry at sulfur. The solvate thiourea:morpholine (4:3) crystallizes in P21/c with Z = 2. The asymmetric unit contains two independent molecules of thiourea, one morpholine on a general position, and one morpholine disordered over an inversion centre. The thiourea molecules combine to form an open framework with a series of channels, in which the morpholine molecules are attached. The solvate thiourea:1,4-dioxane (4:1) crystallizes in P21/n with Z = 2. The asymmetric unit contains two independent molecules of thiourea and one molecule of dioxane across an inversion centre. One thiourea molecule and the dioxane combine to form a layer structure. The second thiourea molecule links these layers in the third dimension.

Bis{2,4-dibromo-6-[(E)-(4-fluorobenzyl)iminomethyl]phenolato-κ2N,O}cobalt(II)

The complete mol­ecule of the title complex, [Co(C14H9Br2FNO)2], is generated by crystallographic twofold symmetry, with the CoII atom lying on the rotation axis. The coordination of the metal atom by the two N,O-bidentate ligands results in a squashed CoN2O2 tetra­hedron. The six-membered chelate ring is an envelope, with the metal atom as the flap. The dihedral angle between the planes of the aromatic rings within each ligand is 84.1 (6)°.

In Silico Modeling of the Molecular Structure and Binding of Leukotriene A4 into Leukotriene A4 Hydrolase

A combined molecular docking and molecular structure in silico analysis on the substrate and product of leukotriene A4 hydrolase (LTA4H) was performed. The molecular structures of the substrate leukotriene A4 (LTA4) and product leukotirene B4 (LTB4) were studied through density functional theory (DFT) calculations at the B3LYP/6-31 + G(d) level of theory in both gas and condensed phases. The whole LTB4 molecule was divided into three fragments (hydrophobic tail, triene motif, and a polar acidic group) that were subjected to a full conformational study employing the most stable conformations of them to build conformers of the complete molecule and geometry optimize further. LTA4 conformers' structures were modeled from the LTB4 minimum energy conformers. Both protonated and deprotonated species of LTA4 and LTB4 were analyzed according to pKa values found in the literature. Finally, a binding model of LTA4 with LTA4 hydrolase is proposed according to docking results that show intermolecular interactions that position the protonated and deprotonated ligand in the active site, in excellent agreement with the model suggested from LTA4H-inhibitors crystallographic data.

2,2′-Diethoxy-4,4′-[(E,E)-hydrazinediylidenebis(methanylylidene)]diphenol

The complete molecule of the title compound, C18H20N2O4, is generated by inversion symmetry. The conformation around the C=N bond is E. With the exception of the eth­oxy substituent, the mol­ecule is essentially planar with an r.m.s. deviation of 0.0455 Å. In the crystal, mol­ecules are linked by O—H⋯N hydrogen bonds into a two-dimensional supra­molecular network parallel to the bc plane.

A novel method for hydrophobin extraction using CO2 foam fractionation system

Due to the exceptional properties and many potential applications of hydrophobins, special fungal proteins, it becomes necessary to develop a real scale procedure for their production and purification. In our previous study (Deckers et al., 2010) [CO2–hydrophobin structures acting as nanobombs in beer, Brew. Sci. 63:54–61], the strong interaction of CO2–hydrophobin was demonstrated. For the first time, in an approach to isolate hydrophobin HFBII from the growth media of Trichoderma reesei, a foam fractionation system using CO2 as the sparging gas was investigated in this study. Using CO2 foam fractionation, the concentration of HFBII was increased from 0.10±0.02mg/mL up to 0.57±0.04mg/mL. This was shown after a purification step by conventional liquid chromatography and identification of the goal protein using MALDI-TOF. The obtained molecular weight of the protein was 7.042kDa which corresponds to the complete molecule of HFBII, minus the last aminoacid. Micro-spectrophotometry was used for quantification of purified HFBII. Moreover, different parameters of the foam fractionation system were optimized. The concentration of the protein after treatment by CO2 followed by liquid chromatography was increased from 0.32±0.02 to 0.44±0.06mg/mL when the flow rate of gas injection was changed in the range of 1–3L/min. The highest amount of HFBII equal to 0.57±0.04mg/mL was obtained by the highest ratio of liquid height over the column height. Using the larger pore size frits causes increased protein absorption as well. The gushing potential of samples revealed that in contrast to the samples before CO2 treatment, interestingly, no gushing was observed for the samples after treatment. The possibility that stable aggregates of HFBII molecules are formed as a consequence of their high concentration is discussed in this paper. By using DLS analysis of the overfoam, 100nm particle size of CO2 nanobubbles coated by HFBII was obtained. The final concentration of the protein was carried out using Amicon® ultracentrifuge device with the average recovery of 63.8±8.2%.

4-Hydroxybenzamide 1,4-dioxane hemisolvate

The asymmetric unit of the title compound, C7H7NO2·0.5C4H8O2, is composed of one 4-hy­droxy­benzamide mol­ecule and half of a 1,4-dioxane mol­ecule. The complete dioxin molecule is generated by crystallographic inversion symmetry. The crystal has an extensive system of hydrogen bonds, in which the three donor H atoms are fully utilized: these result in amide–amide homodimers, and N—H⋯O(dioxane) and O—H⋯O(amide) links.

2,2′-Bithiophene-3,3′-dicarbonitrile

The complete mol­ecule of the title compound, C10H4N2S2, is generated by an inversion center situated at the mid-point of the bridging C—C bond. The bithio­phene ring system is planar [maximum deviation = 0.003 (2) Å] and the central C—C bond length is 1.450 (2) Å. There are no significant inter­molecular inter­actions in the crystal structure, which is stabilized by van der Waals inter­actions.

Diethyl 2,2′-(1,4-phenylenedioxy)diacetate

In the title compound, C14H18O6, a crystallographic center at the centroid of the aromatic ring generates the complete mol­ecule which is planar within 0.085 (1) Å for the non-H atoms. In the crystal, weak C—H⋯O and C—H⋯π inter­actions link the molecules.