Surface-initiated growth of poly d(A-T) by Taq DNA polymerase

In this paper, we report surface-initiated d(A-T) polymerization by Taq DNA polymerase as a method for constructing DNA-tethered surfaces using an enzyme. The enzymatic polymerization was conducted successfully via two steps: tethering of oligo d(A-T)s onto the surface presenting carboxylic acids by amide coupling and surface-initiated polymerization using Taq DNA polymerase. In this enzymatic polymerization process, the design and construction of carboxylic acid-presenting surfaces were found to be an important factor: DNA growth did not occur on the gold surface coated only with the self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA), but effectively proceeded on the surfaces presenting mixed SAMs of MHDA and 1-pentadecanethiol. The coupling of oligo d(A-T)s and the subsequent DNA polymerization reaction were characterized by polarized infrared external reflectance spectroscopy, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy.     

Enantioreversal in the sharpless asymmetric epoxidation reaction controlled by the molecular weight of a covalently appended achiral polymer

Polymers such as poly(ethylene glycol) (PEG) have proven use in a variety of applications including organic synthesis. We now disclose our investigations into the recently disputed report that PEG tartrate esters can reverse the enantioselectivity of the Sharpless asymmetric epoxidation reaction. The results presented herein have clarified that the enantioselectivity of this reaction can be reproducibly reversed solely as a function of the molecular weight of the appended PEG. By preparing a range of tartrate ligands with varying PEG chains lengths, the reversal was found to occur within a molecular weight change of only 800. As the PEG chain did not affect the inherent chirality of the ligand, the enantioreversal was proposed to occur as a result of two Ti-ligand complexes which differ in their molecularity of ligand, one monomeric in ligand and the other dimeric. Support for this hypothesis was given through equilibrium measurements which revealed that the predominant species in Ti/PEG tartrate ester mixtures is a distinct 2:1 Ti-ligand complex, as opposed to the 2:2 Ti-ligand complex of traditional Sharpless asymmetric epoxidations. In total, these data represent an unrecognized property of PEG-supported catalysts that could open up new venues in the control of asymmetric reactions by means of achiral appended polymers.     

De novo synthesis of stable tetrahydroporphyrinic macrocycles: bacteriochlorins and a tetradehydrocorrin

[structures: see text] Bacteriochlorins (tetrahydroporphyrins) are attractive for diverse photochemical applications owing to their strong absorption in the near-infrared spectral region, as exemplified by the bacterial photosynthetic pigment bacteriochlorophyll a, yet often are labile toward dehydrogenation to give the chlorin. Tetradehydrocorrins (ring-contracted tetrahydroporphyrins) are attractive for studies of catalysis analogous to that of vitamin B12. An eight-step synthesis toward such tetrahydroporphyrinic macrocycles begins with p-tolualdehyde and proceeds to a dihydrodipyrrin-acetal (1) bearing a geminal dimethyl group and a p-tolyl substituent. Self-condensation of 1 in CH3CN containing BF3 x OEt2 at room temperature afforded a readily separable mixture of two free base bacteriochlorins and a free base B,D-tetradehydrocorrin. Each bacteriochlorin contains two geminal dimethyl groups to lock-in the bacteriochlorin hydrogenation level, p-tolyl substituents at opposing (2,12) beta-positions, and the absence (H-BC) or presence (MeO-BC) of a methoxy group at the 5- (meso) position. The B,D-tetradehydrocorrin (TDC) lies equidistant between the hydrogenation levels of corrin and corrole, is enantiomeric, and contains two geminal dimethyl groups, 2,12-di-p-tolyl substituents, and an acetal group at the pyrroline-pyrrole junction. Examination of the effect of the concentrations of 1 (2.5-50 mM) and BF3 x OEt2 (10-500 mM) revealed a different response surface for each of H-BC, MeO-BC, and TDC, enabling relatively selective preparation of a given macrocycle. The highest isolated yield of each was 49, 30, and 66%, respectively. The macrocycles are stable to routine handling in light and air. The bacteriochlorins display characteristic spectral features; for example, H-BC exhibits near-IR absorption (lambda(Qy) = 737 nm, epsilon(Qy) = 130,000 M(-1) cm(-1)) and emission (lambda(em) = 744 nm, phi(f) = 0.14). In summary, this simple entry to stable bacteriochlorins and tetradehydrocorrins should facilitate a wide variety of applications.     

Enantioselectivity of 6-O-alkyldimethylsilyl-2,3-di-O-methyl-β-cyclodextrins as chiral stationary phases in capillary GC

Summary Clenbuterol has been determined in urine by solidphase extraction on a C₁₈ cartridge, diazotization of the eluate with nitrite, coupling of the diazonium ion with 1-(naphthyl)ethylenediamine, and separation of the azo dye formed by HPLC with a C₁₈ column and a micellar mobile phase containing 0.1 M sodium dodecyl sulphate, 12%n-butanol and 0.05 M citrate buffer, pH 3. Recoveries higher than 90% were obtained by mixing the samples with a 20% 0.2 M NaOH before extraction. Limits of detection of 51 and 6.7 ng L⁻¹ were obtained with spectrophotometric and thermal lens spectrometric detection, respectively; respective repeatabilities were 3.1% (5 μg mL⁻¹) and 5.6% (0.16 μg mL⁻¹).     

The direct conversion of carbamates to ureas using aluminum amides

The conversion of carbamates into ureas using aluminum amide complexes is reported. This reaction is a convenient method to prepare bi-, tri- and tetra-substituted ureas from carbamate-protected primary or secondary amines by reaction with primary or secondary amines in the presence of stoichometric quantities of trimethylaluminum. A reactivity trend of the various carbamates was observed and methyl and benzyl carbamates were reacted selectively in the presence of t-butyl carbamates.     

The use of a chelating resin column for preconcentration of trace elements from sea-water in their determination by neutron-activation analysis

A Chelex-100 resin column has been employed for the preliminary concentration of trace elements in water samples before their determination by neutron-activation analysis. The column, filled with a 1:1 mixture of the resin (50-100 mesh) and Pyrex glass powder of the same mesh-size, is shown to maintain a constant flow-rate and give reproducible results. By a combination of preconcentration and neutron-activation analysis it is possible to determine Ba, Ca, Cd, Ce, Co, Cr, Cu, Fe, La, Mg, Mn, Sc, U, V and Zn in sea-water and/or fresh water simultaneously at the parts per milliard level.     

(RO)(2)Ta[tris(2-oxy-3,5-dimethylbenzyl)amine]: structure and lactide polymerization activities

The novel atrane-like six-coordinate (RO)(2)TaL complexes [where R = Me or Et and L = tris(2-oxy-3,5-dimethylbenzyl)amine] containing three six-membered rings have been synthesized and characterized. The R = Me complex is the first group 5 representative of this class of compounds structurally characterized by X-ray means. Somewhat surprisingly, these compounds failed to function as single-site initiators for the polymerization of l-LA to isotactic PLA and rac-LA to atactic PLA, whereas Ta(OEt)(5) and two titanium analogues ROTiL (where R = 2,6-di-i-PrC(6)H(3) and i-Pr) as well as Ti(O-i-Pr)(4) were effective catalysts for both polymerizations.     

Kinesin superfamily protein member 4 (KIF4) is localized to midzone and midbody in dividing cells

In association with microtubules, a variety of kinesins play important roles in cellular functions such as intracellular transport of organelles or vesicles, signal transduction, and cell division. In a previous study we revealed that human kinesin superfamily protein member 4 (KIF4) is a chromokinesin that binds to chromosomes. Since localization of several kinds of kinesin at midzone called central spindle, or midbody that connects two daughter cells, or both, suggests their implication in cell division, we investigated KIF4 localization of during mitosis and cytokinesis in Hela cells. In addition to association with segregating chromosomes through entire mitosis, it also localized to the midzone and to midbody at ana/telophase through cytokinesis. Especially in cells at cytokinesis, KIF4 appeared as a doublet facing each other at the apical ends of two daughter cells. Three- dimensional analysis of architectural relationship between microtubule bundles and KIF4 indicated that KIF4 forms a ring structure wrapping around the microtubule bundles. These results suggest that KIF4 is involved in cytokinesis, although direct evidence was not provided in this study.     

Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine beta-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C(18)-bonded stationary phase silica surface.     

In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4(+)CD25(+) regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4(+) and CD8(+) T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4(+) or CD8(+) T cells with a CD4(+) or CD8(+) T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.