Recent progress in the development of derivatization reagents having a benzofurazan structure

Chemical derivatization is often used to enhance the detectability of the target compounds and to improve the separation efficiency in high-performance liquid chromatography (HPLC). In this review, we describe the recent progress in the development of derivatization reagents having a benzofurazan structure, namely, the fluorogenic reagents, water-soluble reagents, reagents for the analysis of peptides and proteins, and reagents for mass spectrometric detection. The application of these reagents to bio-samples is also briefly described.     

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.     

Versatile site-specific conjugation of small molecules to siRNA using click chemistry

We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5'-alkyne moieties were used for conjugation to the 5'-end of the oligonucleotide. Previously described 2'- and 3'-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3' and 5' termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.     

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.     

Photosynthetic oxygen evolution in mesoporous silica material: adsorption of photosystem II reaction center complex into 23 nm nanopores in SBA

An oxygen-evolving photosynthetic reaction center complex (PSII) was adsorbed into nanopores in SBA, a mesoporous silica compound. We purified the dimer of PSII complex from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, which grows optimally at 57 °C. The thermally stable PSII dimeric complex has a diameter of 20 nm and a molecular mass of 756 kDa and binds more than 60 chlorophylls. The SBA particles, with average internal pore diameters of 15 nm (SBA(15)) and 23 nm (SBA(23)), adsorbed 4.7 and 15 mg of PSII/g SBA, respectively. Measurement with a confocal laser-scanning microscope indicated the adsorption of PSII to the surface and the inner space of the SBA(23) particles, indicating the adsorption of PSII into the 23 nm silica nanopores. PSII did not bind to the inner pores of SBA(15). PSII bound to SBA(23) showed the high and stable activity of a photosynthetic oxygen-evolving reaction, indicating the light-driven electron transport from water to the quinone molecules added in the outer medium. The PSII-SBA conjugate can be a new material for photosensors and artificial photosynthetic systems.     

Preparation and optical and electrochemical properties of octaethylphthalocyanines fused with several tetrathiafulvalene units

3,6‐Diethylphthalonitrile ( 3 ) with a tetrathiafulvalene (TTF) unit at 4,5‐positions was prepared from 4,5‐xylylenedithio‐3,6‐diethylphthalonitrile ( 1a ) via elimination of the xylylene group, connection of a carbonyl group to benzenedithiolate generated, and condensation of 4,5‐bis(methylthio)‐1,3‐dithiole‐2‐thione with benzo‐1,3‐dithiole‐2‐one ( 2‐O ) produced. A 1:1 mixture of phthalonitrile ( 3 ) and 4,5‐bis(benzylthio)‐3,6‐diethylphthalonitrile ( 1b ) was treated with lithium in n‐hexanol at 120°C to produce hexakis (benzylthio)mono(tetrathiafulvaleno)phthalocyanine ( 5 ), tetrakis(benzylthio)bis(tetrathiafulvaleno)phthalocyanine ( 6 ), and bis(benzylthio)tris(tetrathiafulvaleno)phthalocyanine ( 7 ). The structures of 5 , 6 , and 7 were determined by ¹H NMR, FAB MS, MALDI‐TOF MS (matrix assisted laser desorption ionization time‐of‐flight mass spectrometry), and UV‐‐vis spectroscopy. Compound 6 is a mixture of trans and cis isomers ( 6‐ trans and 6‐ cis ). The UV‐‐vis spectrum of 5 measured in chloroform changed by addition of trifluoroacetic acid (TFA). The Q band absorption at λ_max = 755 nm (chloroform) decreased in intensity and resulted in a new absorption at λ_max = 740 nm (chloroform/TFA). The electrochemical properties of 5 , 6 , and 7 were determined by cyclic voltammetry using Ag/AgNO₃ as a reference electrode. © 2011 Wiley Periodicals, Inc. Heteroatom Chem 22:605–611, 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.20694     

Excited-state and photoelectrochemical behavior of pyrene-linked phenyleneethynylene oligomer

An oligophenyleneethynylene (OPE), 1,4-bis(phenyleneethynyl)-2,5-bis(hexyloxy)benzene (2), is coupled with pyrene to extend the conjugation and allow its use as a light-harvesting molecule [Py-OPE (1)]. The absorption and emission maxima of 1 are red-shifted compared to those of 2. Similar differences in the singlet and triplet excited-state properties are evident. The fluorescence yield of 2 in toluene is 0.53, which is slightly less than the value for the parent OPE (2) of 0.66. The excited singlet and triplet of 1 as characterized from transient absorption spectroscopy exhibit lifetimes of 1.07 ns and 4.0 micros, respectively, in toluene. When 1 was cast as a film on a glass electrode (OTE) and excited with a 387-nm laser pulse, we observed the formation of excitons that decayed within a few picoseconds. When 1 was cast as a film on a SnO2-modified conducting glass electrode (OTE/SnO2), a small fraction of excitons dissociated to produce a long-lived charge-separated state. The role of the SnO2 interface in promoting charge separation was inferred from the photoelectrochemical measurements. Under visible light excitation, the OTE/SnO2 electrode was capable of generating photocurrent (approximately 0.25 mA/cm2) with an incident photon conversion efficiency (IPCE) of approximately 6%.     

Hexagonal-close-packed, hierarchical amorphous TiO2 nanocolumn arrays: transferability, enhanced photocatalytic activity, and superamphiphilicity without UV irradiation

A hexagonal-close-packed (hcp), hierarchical amorphous TiO2 nanocolumn array was fabricated by pulsed laser deposition (PLD) using a PS colloidal monolayer as a template under a high pressure (6.7 Pa) of background oxygen gas. The formation mechanism was investigated, and a model of multidirection glancing deposition was proposed to explain the formation process. This strategy can be extended to the fabrication of similar structures using different materials. Interestingly, this nanostructured array could be transferred to almost any substrate, avoiding restriction of substrate types in fabrication of nanocolumn arrays, which is helpful in the design and creation of nanodevices on various desired substrates. This hierarchical nanocolumn array exhibits excellent superamphiphilicity with both water and oil contact angles of 0 degrees, without further UV irradiation. More importantly, the amorphous TiO2 nanocolumn array demonstrates better performance in photocatalytic activity than an anatase nanocolumn array due to its large surface area and special microstructures, suggesting that the surface area of the TiO2 is preferable to its crystal structure for enhancing photocatalytic activity. The combination of superamphiphilicity and photocatalytic activity gives the surface an excellent self-cleaning effect.     

A novel transformation of 1-exo-substituted 2a-aroyl-1,2,2a,8b-tetrahydro-3H-benzo[b]cyclobuta[d]pyran-3-ones with sulfoxonium ylide to highly strained 2a-(1-arylethenyl)-1,2,2a,7b-tetrahydrocyclobuta[b]benzofurans

When 1-substituted 2a-aroyl-1,2,2a,8b-tetrahydro-3H-benzo[b]cyclobuta[d]pyran-3-ones (1) were treated with dimethylsulfoxonium methylide, 1-endo isomers (endo-1) gave 1-substituted 3-aroyl-1,2,4a,9b-tetrahydrodibenzofuran-4-ols (2) exclusively as expected. On the other hand, 1-exo isomers (exo-1) underwent a novel transformation to 1-substituted 2a-(1-arylethenyl)-1,2,2a,7b-tetrahydrocyclobuta[b]benzofurans (3), together with 2.     

Reactions of Mo(II)-tetraphosphine complex [MoCl2{meso-o-C6H4(PPhCH2CH2PPh2)2}] with a series of small molecules

Reactions of Mo(II)-tetraphosphine complex [MoCl₂(κ⁴-P4)] (2; P4 = meso-o-C₆H₄(PPhCH₂CH₂PPh₂)₂) with a series of small molecules have been investigated. Thus, treatment of 2 with alkynes RCCR′ (R = Ph, R′ = H; R = p-tolyl, R′ = H; R = Me, R′ = Ph) in benzene or toluene gave neutral mono(alkyne) complexes [MoCl₂(RCCR′)(κ³-P4)] containing tridentate P4 ligand, which were converted to cationic complexes [MoCl(RCCR′)(κ⁴-P4)]Cl having tetradentate P4 ligand upon dissolution into CDCl₃ or CD₂Cl₂. The latter complexes were available directly from the reactions of 2 with the alkynes in CH₂Cl₂. On the other hand, treatment of 2 with 1 equiv. of XyNC (Xy = 2,6-Me₂C₆H₃) afforded a seven-coordinate mono(isocyanide) complex [MoCl₂(XyNC)(κ⁴-P4)] (7), which reacted further with XyNC to give a cationic bis(isocyanide) complex [MoCl(XyNC)₂(κ⁴-P4)]Cl (8). From the reaction of 2 with CO, a mono(carbonyl) complex [MoCl₂(CO)(κ⁴-P4)] (9) was obtained as a sole isolable product. Reaction of 9 with XyNC afforded [MoCl(CO)(XyNC)(κ⁴-P4)]Cl (10a) having a pentagonal-bipyramidal geometry with axial CO and XyNC ligands, whereas that of 7 with CO resulted in the formation of a mixture of 10a and its isomer 10b containing axial CO and Cl ligands. Structures of 7 and 9 as well as [MoCl(XyNC)₂(κ⁴-P4)][PF₆](8′) and [MoCl(CO)(XyNC)(κ⁴-P4)][PF₆] (10a′) derived by the anion metathesis from 8 and 10a, respectively, were determined in detail by the X-ray crystallography.