TNA synthesis by DNA polymerases

Threose nucleic acid (TNA), which has a repeat unit one atom shorter than that of DNA, is capable of Watson-Crick base pairing with DNA, RNA, and TNA. Because of its chemical simplicity, TNA is considered to be a possible progenitor of RNA. As an initial step toward developing the molecular tools necessary to investigate the functional capabilities of TNA by in vitro selection, we have screened a variety of DNA polymerases for TNA synthesis activity on a DNA template. We wish to report that several polymerases show surprisingly good ability to synthesize TNA using alpha-l-threofuranosyl thymidine-3'-triphosphate as a substrate.     

Characterization of clean and oxidized (100)LaB6

We have studied clean and oxidized (100)LaB₆ grown from aluminum melts by making Auger, LEED, evaporation and work function measurements on well-defined surfaces. The clean surface shows no La enrichment when initially heated as high as 1700°C. Its LEED pattern is 1 × 1, indicating no surface reconstruction. Langmuir evaporation studies up to temperatures of 1700°C show only La and B evaporating non-congruently, and LaO. The activation energy for B evaporation from LaB₆ (and from CeB₆ and EuB₆ also) is abot 5.5 eV, very close to that from elemental B. The rare-earth activation energies, however, vary, being highest for the rare-earth whose pure metal vapor pressure is lowest. Oxidation was carried out at room temperature using O₂ pressures up to 10^?7 Torr and at 1000°C using O₂ pressures up to 10^?4 Torr. At room temperature oxygen adsorption proceeeds to a saturated value indicated from LEED behavior to be about one monolayer. It produces a monotonic work function increase, which also saturates (at 1.40 V), varying linearly with the oxygen uptake. Oxidation at 1000°C is much more extensive than at room temperature, involving at least several monolayers, and results in a work function increase of 2.42 V. Results are discussed in terms of a terminal plane composed of La atoms, and adsorbed oxygen which, when given sufficient mobility, prefers bonding to La atoms at sites atop the B octahedra.     

Low-temperature photoluminescence of p-nitroaniline and o-methyl-p-nitroaniline crystals

Photoemission spectra of para-nitroaniline (p-NA) and ortho-methyl-para-nitroaniline (MNA) single crystals measured between 5 and 250K revealed that the MNA crystals emit phosphorescence and fluorescence in contrast with the p-NA crystals which have only fluorescence. It is assumed that the fluorescence of p-NA crystals at 5K originates from the doublet state of negatively charged polarons while the fluorescence of MNA crystals at 60K is due to trap states. The Stokes shift between absorption and emission onsets, measured at 5K, is much larger in the p-NA crystals than in the MNA and meta-nitroaniline (m-NA) crystals. This fact is rationalized by different geometrical changes caused by excitation.     

Low-temperature phase transition in [Mn(OS(CH3)2)6](ClO4)2 studied by single crystal X-ray diffraction, infrared absorption and Raman scattering spectroscopies

Single crystal X-ray diffraction studies of [Mn(OS(CH3)2)6](ClO4)2 have shown that the low temperature phase transition, detected by differential scanning calorimetry (DSC) at about 223 K, is associated with the crystal symmetry's reduction from an orthorhombic crystallographic system (Fdd2, No. 43) to a monoclinic one (Cc, No. 9). The analysis of the full width at half maximum of the bands connected with: δd(OClO)F2 and ρ(CH3) vibrational modes in the FT-IR and FT-RS spectra, respectively, registered in the function of temperature, proved that the reorientational motions of ClO4- anions and CH3 groups from (CH3)2SO ligands, began to slow down at temperatures below the phase transition at about 223K. Mean values of activation energy for ClO4- reorientation in the high temperature phase I and low temperature phase II are: Ea(I)≈14 kJ mol(-1) and Ea(II)≈10 kJ mol(-1), respectively. Analogous values for CH3 reorientation are: Ea(I)≈23 kJ mol(-1) and Ea(II)≈1 kJ mol(-1), respectively.     

Phase polymorphism of [Zn(DMSO)6](ClO4)2 studied by differential scanning calorimetry

Four solid phases of [Zn(DMSO)₆](ClO₄)₂ have been detected by differential scanning calorimetry (DSC). Specifically, the phase transitions were detected between: metastable phase KII ↔ supercooled phase K0 at , stable phase KIb ↔ stable phase KIa at , stable phase KIa ↔ stable phase K0 at . At T_m2 = 389 K crystals partially and at T_m1 = 465 K completely melts. From the entropy change values it was concluded that the phases: K0 and K0′ are the orientationally dynamically disordered phases, so called ODDIC crystals, and phases KIa, KIb and metastable KII are dynamically ordered but with some degree of positional disorder.     

Glycerol nucleoside triphosphates: synthesis and polymerase substrate activities

[Structure: see text] The synthesis of (S)-glycerol nucleoside triphosphates (gNTPs) and the analysis of their substrate activities for enzymatic polymerization is described. NTPs with simplified carbohydrate backbones such as the tNTPs (alpha-L-threose-NTPs) are polymerase substrates and offer the potential to create non-natural aptamer sequences with simplified backbones through enzymatic means. The acyclic (S)-GNA was modeled after the shortened alpha-threofuranosyl backbone. Here we describe the synthesis of (S)-glycerol NTPs and initial enzymatic testing of this further simplified nucleic acid backbone.     

Aptamers selected for higher-affinity binding are not more specific for the target ligand

Previous study of eleven different in vitro-selected RNA aptamers that bind guanosine triphosphate (GTP) with K(d)s ranging from 8 microM to 9 nM showed that more information is required to specify the structures of the higher-affinity aptamers. We are interested in understanding how the more complex aptamers achieve higher affinities for the ligand. In vitro selection produces structural solutions to a functional problem that are are as simple as possible in terms of the information content needed to define them. It has long been assumed that the simplest way to improve the affinity of an aptamer is to increase the shape and functional group complementarity of the RNA binding pocket for the ligand. This argument underlies the hypothesis that selection for higher-affinity aptamers automatically leads to structures that bind more specifically to the target molecule. Here, we examined the binding specificities of the eleven GTP aptamers by carrying out competition binding studies with sixteen different chemical analogues of GTP. The aptamers have distinct patterns of specificity, implying that each RNA is a structurally unique solution to the problem of GTP binding. However, these experiments failed to provide evidence that higher-affinity aptamers bind more specifically to GTP. We suggest that the simplest way to improve aptamer K(d)s may be to increase the stability of the RNA tertiary structure with additional intramolecular RNA-RNA interactions; increasingly specific ligand binding may emerge only in response to direct selection for specificity.     

IPr# – highly hindered,broadly applicable N-heterocyclic carbenes

IPr (IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) represents the most important NHC (NHC = N-heterocyclic carbene) ligand throughout the field of homogeneous catalysis. Herein, we report the synthesis, catalytic activity, and full structural and electronic characterization of novel, sterically-bulky, easily-accessible NHC ligands based on the hash peralkylation concept, including IPr#, Np# and BIAN-IPr#. The new ligands have been commercialized in collaboration with Millipore Sigma: IPr#HCl, 915653; Np#HCl; 915912; BIAN-IPr#HCl, 916420, enabling broad access of the academic and industrial researchers to new ligands for reaction optimization and screening. In particular, the synthesis of IPr# hinges upon cost-effective, modular alkylation of aniline, an industrial chemical that is available in bulk. The generality of this approach in ligand design is demonstrated through facile synthesis of BIAN-IPr# and Np#, two ligands that differ in steric properties and N-wingtip arrangement. The broad activity in various cross-coupling reactions in an array of N–C, O–C, C–Cl, C–Br, C–S and C–H bond cross-couplings is demonstrated. The evaluation of steric, electron-donating and π-accepting properties as well as coordination chemistry to Au(i), Rh(i) and Pd(ii) is presented. Given the tremendous importance of NHC ligands in homogenous catalysis, we expect that this new class of NHCs will find rapid and widespread application.

We report novel, sterically-bulky, easily-accessible NHC ligands based on the hash peralkylation concept. The new ligands have been commercialized in collaboration with Millipore Sigma: IPr#HCl, 915653; Np#HCl; 915912; BIAN-IPr#HCl, 916420.