Phosphoproteome Profiling Using a Fluorescent Phosphosensor Dye in Two-Dimensional Polyacrylamide Gel Electrophoresis

We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4–5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.     

Anomalous dielectric behavior and thermal motion of water molecules confined in channels of porous coordination polymer crystals

Guest water molecules confined in channels of porous coordination polymer crystals [Ln(2)Cu(3)(IDA)(6)]·nH(2)O (Ln = La, Nd, Sm, Gd, Ho, Er; IDA = [NH(CH(2)COO)(2)](2-); n ≈ 9) exhibited large dielectric constants (ε) and antiferroelectric behaviors at high temperatures (e.g., ε(Sm) ≈ 1300 at 400 K). In addition, plots of the temperature dependence of ε showed broad peaks at ～170 K, below which ε became very small. These puzzling temperature dependences of ε are consistent with the results of molecular dynamics simulations, suggesting the "freezing of thermal motion" of water molecules at ～170 K.     

Peroxidase activity enhancement of horse cytochrome c by dimerization

The peroxidase activity of horse cytochrome c was enhanced by its dimerization, where its Compound III (oxy-form) and Compound I (oxoferryl porphyrin π-cation radical) species were detected in the reactions with hydrogen peroxide and meta-chloroperbenzoic acid, respectively. These results show that oligomeric cytochrome c can contribute as a proapoptotic conformer by the increased peroxidase activity.     

Synthesis of the ABC ring system of Jiadifenin via Pd-catalyzed cyclizations

An efficient route toward the central ABC system of jiadifenin has been developed using two key Pd-catalyzed cyclizations. A protic solvent-activated Mizoroki-Heck reaction was used to construct the C(9) quaternary carbon and the A ring. A cascading Tsuji-Trost cyclization/lactonization sequence was employed to establish the BC ring system and the C(5,6) stereochemistry.     

Cooperative catalytic reactions using organocatalysts and transition metal catalysts: enantioselective propargylic alkylation of propargylic esters with aldehydes

The enantioselective propargylic alkylation of propargylic esters with aldehydes in the presence of a copper complex and an optically active secondary amine as cocatalysts has been found to give the corresponding propargylic alkylated products in good yields as a mixture of two diastereoisomers with a high enantioselectivity.     

Copper-catalyzed enantioselective propargylic amination of nonaromatic propargylic esters with amines

The enantioselective propargylic amination of propargylic pentafluorobenzoates bearing an alkyl group at the propargylic position with amines in the presence of catalytic amounts of a copper complex and an optically active diphosphine such as BINAP has been found to give the corresponding propargylic amines in good yields with high enantioselectivity.     

On-chip fraction collection for multiple selected ssDNA fragments using isolated extraction channels

High efficiency and high-purity fraction collection is highly sought in analysis of fragments-of-interest from selective polymerase chain reaction (PCR) products generated by High Coverage Gene Expression Profiling (HiCEP) methods. Here we demonstrate a new electrophoretic chip device enabling automatic high-efficient fractionation of multiple ssDNA target fragments during a run of separation. We used thoroughly isolated extraction channels for each selected target to reduce the risk of cross-contamination between targets due to cross-talk of extraction channels. Fragments of 35, 108 and 138 b, were successfully isolated, then the recovery was PCR-amplified and assessed by capillary electrophoresis (CE) analysis. Total impurity level of the targets due to unwanted fragments of 0.7%, 2% and 6% respectively, was estimated. Difficulties in collecting multiple target factions are due to band diffusion and DNA adsorption to the walls for the fragments in the separation channel, which is generated by transferring the DNA target fraction from the extraction section to the target reservoir. Therefore, we have carefully measured band broadening and analyzed its influence on the separation resolution due to the delay.     

Design and stereoselective synthesis of four peptide nucleic acid monomers with cyclic structures in backbone

Four isomers of the monomer of peptide nucleic acid (PNA) were derived from (2S,4R)‐4‐hydroxyproline; they had different stereochemistries at the C² and C⁴ positions in the pyrrolidine ring. These different backbone conformations corresponding to four different stereochemistries were realized through a combination of inversions at the C² and the C⁴ positions in pyrrolidine ring. The obtained backbone frameworks were reacted with N‐benzoyl thymine to give the corresponding PNA monomers. Spectroscopic comparison of the resultant monomers confirmed their stereochemistries. J. Heterocyclic Chem., (2011).     

Stereochemically controlled asymmetric 1,2-reduction of enones mediated by a chiral sulfoxide moiety and a lanthanum(III) ion

Enantiomerically pure (Z)-β-sulfinyl allylic alcohols of either handedness can be readily prepared from (Z)-β-sulfinyl enones using NaBH(4) or DIBAL reductants in the presence of LaCl(3) as a chelating agent. A chiral sulfoxide auxiliary induces the remote 1,2-asymmetric reduction (1,4-induction) to afford various chiral allylic alcohols in high yields with excellent stereoselectivities (up to 100% de).     

Size-dependent inclusion of organic molecules into elastic pillared carbons

Size selective inclusion of organic molecules into pillared carbons accompanied by large interlayer expansions was observed for the first time. In order to achieve this, the distance between two adjacent pillars should be between 0.36 and 0.40 nm and they should align in the same direction.