Pd(2+)-exchanged graphite oxide and chemically derived graphenes therefrom were employed as supports for Pd nanoparticles. The influence of catalyst preparation, carbon functionalization, and catalyst morphology on the catalytic activity in the Suzuki-Miyaura coupling reactions was investigated. The catalysts were characterized by means of spectroscopy (FT-IR, solid-state (13)C NMR, AAS, XPS), X-ray scattering (WAXS), surface area analysis (BET, methylene blue adsorption), and electron microscopy (TEM, ESEM). In contrast to the conventional Pd/C catalyst, graphite oxide and graphene-based catalysts gave much higher activities with turnover frequencies exceeding 39,000 h(-1), accompanied by very low palladium leaching (<1 ppm).

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In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

A general chemical phosphorylation method based on P(II1) chemistry has been developed. The system is demonstrated for the phosphorylation of oligonucleotides, directly after their synthesis on a solid support, and for the 0-phosphorylation of serine, threonine, and tyrosine as well as for a serine-containing peptide.

The shift of paradigm in combinatorial chemistry, from large compound libraries (of mixtures) on a small scale towards defined compound libraries where each compound is prepared in an individual well, has stimulated the search for alternative separation approaches. The key to a rapid and efficient synthesis is not only the parallel arrangement of reactions, but simple work-up procedures so as to circumvent time-consuming and laborious purification steps. During the initial development stages of combinatorial synthesis it was believed that rational synthesis of individual compounds could only be achieved by solid-phase strategies. However, there are a number of problems in solid-phase chemistry: most notably there is the need for a suitable linker unit, the limitation of the reaction conditions to certain solvents and reagents, and the heterogeneous reaction conditions. Further disadvantages are: the moderate loading capacities of the polymeric support and the limited stability of the solid support. In the last few years several new separation techniques have been developed. Depending on the chemical problem or the class of compounds to be prepared, one can choose from a whole array of different approaches. Most of these modern separation approaches rely on solution-phase chemistry, even though some of them use solid-phase resins as tools (for example, as scavengers). Several of these separation techniques are based on liquid-liquid phase separation, including ionic liquids, fluorous phases, and supercritical solvents. Besides being benign with respect to their environmental aspects, they also show a number of advantages with respect to the work-up procedures of organic reactions as well as simplicity in the isolation of products. Another set of separation strategies involves polymeric supports (for example, as scavengers or for cyclative cleavage), either as solid phases or as soluble polymeric supports. In contrast to solid-phase resins, soluble polymeric supports allow reactions to be performed under homogeneous conditions, which can be an important factor in catalysis. At the same time, a whole set of techniques has been developed for the separation of these soluble polymeric supports from small target molecules. Finally, miscellaneous separation techniques, such as phase-switchable tags for precipitation by chemical modification or magnetic beads, can accelerate the separation of compounds in a parallel format.

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70' (Mr 70000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were asoated with p70" activation, the kinase was labeled to high specific activity with 32P1 in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensonal tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide ofMr 2400. Analysis of the p70"" protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vvo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p7O"". In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.Growth factors induce quiescent cells in culture to reenter the cell cycle, replicate their DNA, and divide through a complex array ofbiochemical responses (1). In most cases this process is initiated through specific ligand-activated receptor tyrosine kinases at the cell surface but is orchestrated intracellularly by a network of activated serine/threonine kinases (2). The mechanisms by which tyrosine kinases activate serine/ threonine kinases are still unclear, with the possible exception of protein kinase C (2, 3). One of the many early obligatory responses elicited by growth factors is the activation and maintenance of high rates of protein synthesis, which is required for reentry and transit through the G1 phase of the cell cycle (4, 5). This event is associated with increased phosphorylation of serine/threonine residues in a number of specific translational components, including 40S ribosomal protein S6 (6, 7). In vitro and in vivo studies have indicated that phosphorylation at five serine residues at the carboxyl terminus of S6 is required for the activation of protein synthesis (8-11). The kinase mediating this event was first detected in extracts of quiescent cells stimulated to proliferate by serum or epidermal growth factor (12, 13). Purification of this S6 kinase activity eventually revealed an enzyme of Mr 70,000, referred to as p70s6k (14,15), which is highly specific for ribosomal protein S6 (14), biphasically activated (16, 17), and selectively inactivated by type 2A phosphatase (18). The p70s"s phosphorylates four and possibly all five ofthe S6 sites observed in vivo (19), with recognition of the substrate absolutely dependent o...

The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells. CD4+ and CD8+ T cells recognize antigens on major histocompatibility complex (MHC) class II-bearing and class I-bearing target cells respectively. The ability of monoclonal antibodies against CD4 and CD8 to block antigen recognition by T cells, as well as cell-cell adhesion assays, indicate that CD4 and CD8 bind to nonpolymorphic determinants of class II or class I MHC. Here we demonstrate that soluble recombinant HLA-DR4 molecules from insect cells and HLA-DR-derived peptides bind to immobilized recombinant soluble CD4. CD4 binds recombinant soluble DR4 heterodimers, as well as the soluble DR4-beta chain alone. Furthermore, two out of twelve DR4-beta peptides could interact specifically with CD4. These findings show that CD4 interacts with a region of MHC class II molecules analogous to a previously identified loop in class I MHC proteins that binds CD8 (refs 8, 9).