A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4′-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) perstep at 50°C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 × 10¹⁰ strands/mm². This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 × 10¹⁰/mm² and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4′-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.     

On the dissolution processes of Na2I+ and Na3I2+ with the association of water molecules: mechanistic and energetic details

The sequential association energies for one through six water molecules clustering to Na(2)I(+), as well as one and two water molecules clustering to Na(3)I(2)(+), are measured. The association energies show a pairwise behavior, indicating a symmetric association of water molecules to the linear Na(2)I(+) and Na(3)I(2)(+) ions. This pairwise behavior is well reproduced by Density Functional Theory (DFT) calculations. DFT calculations also suggest that a significant separation of charge for the Na-I ion pair occurs when four or more water molecules cluster to a single sodium center. Two different solvent-separated ion pairs have been identified with the DFT calculations. Experiments also show that the dissolution processes, loss of a neutral NaI unit, occurs when six or more water molecules have been added to Na(2)I(+) cluster. However, one or two water molecules are able to detach an NaI unit from the Na(3)I(2)(+) cluster. The difference in solubility of the Na(2)I(+) and Na(3)I(2)(+) ions is due to the difference in the energies required to lose an NaI unit from these two species. The experiment also confirms that the loss of a neutral NaI unit, instead of an Na(+) ion, occurs during the dissolution processes of Na(3)I(2)(+). The microsolvation schemes proposed to explain our experimental observations are supported by DFT and phase space theory (PST) calculations.     

Kinetic and mechanistic studies of the reaction of a range of bases and metal-hydroxo complexes with the phosphonate ester 2,4-dinitrophenyl ethyl methylphosphonate in aqueous solution

The base hydrolysis and spontaneous water hydrolysis of the phosphonate ester 2,4-dinitrophenyl ethyl methylphosphonate (DNPEMP) has been studied in detail and the activation parameters for these processes determined. The catalytic effect of a series of 19 oxygen and nitrogen bases and metal hydroxo complexes has been studied. The Brønsted plot of pK versus logkN has a slope (β) of 0.47, which is fairly typical for phosphate and phosphonate esters. alpha-Effect nucleophiles such as 2-iodosobenzoate, hypochlorite and the hydroperoxide ion (HO-2) exhibit enhanced reactivity, as does fluoride ion and the bifunctional metal complex [Cu(tmen) (OH2)(OH)]+ (tmen = N,N,N',N',-tetramethylethylenediamine). Reactions involving these species appear to take place by a nucleophilic pathway, while the other oxygen and nitrogen bases act as general base catalysts. The solvent deuterium isotope effect for Et3N catalysis, kH2O/kD2O=1.3, is consistent with general base catalysis. The mechanisms of these reactions are discussed in detail.     

An evaluation of exogenous application of protoporphyrin IX and its dimethyl ester as a photodynamic diagnostic agent in poorly differentiated human nasopharyngeal carcinoma

5-Aminolevulinic acid and its esterified analogues have been under much investigation to enhance the endogenous production of protoporphyrin IX (PpIX) in tumor cells. However, in this work, we studied the in vitro and in vivo efficacy of exogenously administered PpIX and its esterified analogue, PpIX dimethyl ester (PME), in poorly differentiated human nasopharyngeal carcinoma (NPC/CNE-2) as a photodynamic diagnostic (PDD) agent. NPC/CNE-2 at its earliest time, 1 h after incubation with PME in in vitro studies, has exhibited 64% (P <0.01) higher tumor to normal cell (T/N) fluorescence ratio than with PpIX. In an in vivo mouse xenograft model, comparable photosensitizer concentration in tumor after intravenous administration was observed at 1-3 h time points, but at 9 h, PME had 31% (P=0.05) greater concentration in tumor compared with PpIX. In addition, by constituting PME and PpIX in different topical gel composites, of which, PME gel composition of 8:2 Plasdone and Gantrez resulted in the highest T/N ratio at 6 h after application (34%; P <0.05) in comparison with other gel composites. Evaluation of PME and PpIX constituted in the delivery vehicles investigated showed comparable selectivity for tumor at 1-3 h, thus neither photosensitizer is more efficient than the other for PDD at the early time points; however, beyond 6 h, PME had higher selectivity for tumor compared with PpIX. Thus, further investigation is warranted to improve the drug delivery vehicle for greater tumor selectivity at a shorter incubation time.     

Artificial multiple criticality and phase equilibria: an investigation of the PC-SAFT approach

The perturbed-chain statistical associating fluid theory (PC-SAFT) is studied for a wide range of temperature, T, pressure, p, and (effective) chain length, m, to establish the generic phase diagram of polymers according to this theory. In addition to the expected gas-liquid coexistence, two additional phase separations are found, termed "gas-gas" equilibrium (at very low densities) and "liquid-liquid" equilibrium (at densities where the system is expected to be solid already). These phase separations imply that in one-component polymer systems three critical points occur, as well as equilibria of three fluid phases at triple points. However, Monte Carlo simulations of the corresponding system yield no trace of the gas-gas and liquid-liquid equilibria, and we conclude that the latter are just artefacts of the PC-SAFT approach. Using PC-SAFT to correlate data for polybutadiene melts, we suggest that discrepancies in modelling the polymer density at ambient temperature and high pressure can be related to the presumably artificial liquid-liquid phase separation at lower temperatures. Thus, particular care is needed in engineering applications of the PC-SAFT theory that aims at predicting properties of macromolecular materials.     

Nonlinear decrease of background fluorescence in polymer thin-films - a survey of materials and how they can complicate fluorescence detection in microTAS

Polymers and plastics are receiving increased attention as materials for microfluidics and microTAS applications. Given the ubiquity of fluorescence detection techniques in micro-analytical systems, the fluorescence properties of polymers and plastics should not be overlooked. We survey some commonly available polymer thin-films for their fluorescence behaviour under standardized conditions to determine which materials are most suitable for high-sensitivity fluorescence detection lab chips. The initial fluorescence intensities of some of the materials surveyed were significantly higher than glass and fused silica controls, and decreased over the three hour period with complex kinetics. We then discuss how this has confounded fluorescence detection in our analytical context, and possible mechanisms for the decrease.     

Ab initio study of the binding of Trichostatin A (TSA) in the active site of histone deacetylase like protein (HDLP)

Histone deacetylase (HDAC) inhibitors have recently attracted considerable interest because of their therapeutic potential for the treatment of cell proliferative diseases. An X-ray structure of a very potent inhibitor, Trichostatin A (TSA), bound to HDLP (an HDAC analogue isolated from Aquifex aeolicus), is available. From this structure, an active site model (322 atoms), relevant for the binding of TSA and structural analogues, has been derived, and TSA has been minimized in this active site at HF 3-21G* level. The resulting conformation is in excellent accordance with the X-ray structure, and indicates a deprotonation of the hydroxamic acid in TSA by His 131. Also, a water molecule was minimized in the active site. In addition to a similar deprotonation, in accordance with a possible catalytic mechanism of HDAC as proposed by Finnin et al. (M. S. Finnin, J. R. Donigian, A. Cohen, V. M. Richon, R. A. Rifkind and P. A. Marks, Nature, 1999, 401, 188-193), a displacement of the resulting OH- ion in the active site was observed. Based on these results, the difference in energy of binding between TSA and water was calculated. The resulting value is realistic in respect to experimental binding affinities. Furthermore, the mechanism of action of the His 131-Asp 166 charge relay system was investigated. Although the Asp residue in this motif is known to substantially increase the basicity of the His residue, no proton transfer from His 131 to Asp 166 was observed on binding of TSA or water. However, in the empty protonated active site, this proton transfer does occur.     

Challenges in the structural characterization of thin organic films

This review addresses the special problems associated with the micro-structural characterization of thin and ultrathin organic films, primarily by optical spectroscopies. Films which are deposited by Langmuir-Blodgett techniques, self-assembly chemistry, and bulk film deposition techniques are considered. The use of enhanced optical excitation using surface phasma resonances and integrated optical structures is discussed extensively, as is the use of ellipsometry. Discussion of the spectroscopies used is broken into a section on electronic spectroscopies and an extensive discussion of vibrational spectroscopies. Vibrational information may be obtained with photons (absorption or scattering) or electrons (loss spectra), and the types of experimental systems amenable to each, along with the advantages and disadvantages of each are explored.     

Conformational preferences of 34 valence electron A2X4 molecules: An ab initio Study of B2F4, B2Cl4, N2O4, and C2O

Ab initio molecular orbital structures and energies of B₂F₄, B₂Cl₄, N₂O₄, and C₂O have been calculated for both perpendicular D_2d and planar D_2h rotamers. The experimental trend toward greater preference for the D_2d forms in going from B₂F₄ to B₂Cl₄ is reproduced. N₂O₄ favors the planar conformation, although the rotation barrier is overestimated at the theoretical levels used. The oxalate dianion is calculated to be more stable in the D_2d conformation; the experimental planar arrangement in the solid may be due to crystal packing forces. The preferences for one conformation over another are small; analysis indicates that different effects may predominate in each case: π stabilization for B₂F₄, hyperconjugation for B₂Cl₄, lone-pair interactions for N₂O₄, and electrostatic repulsions for C₂O.     

Reactions of tetrahydro-as-triazine-3(2H)-thiories with α, β-difunctional compounds

The results of allowing tetrahydro-as-triazine-3(2H)-thiones to react with various α,β-diiunctional compounds, such as, α-bromoethyl p-toluenesulfonate, chloroacetaldehyde, α-bromophenylacet-uldehyde, phenaeyl bromide, chloroacetonitrile, α-bromophenylaeelonitrile, and α-cyanobenzyl p-toluenesulfonate are discussed. These condensations give either a 5H-thiazolo[3,2-b]-as-triazine or a 2H-thiazolo[2,3-c]-as-triazine.