Synthesis and Macrodomain Binding of Mono‐ADP‐Ribosylated Peptides

Mono‐ADP‐ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono‐ADP‐ribosylated proteins have been described. We report the synthesis of ADP‐ribosylated peptides from the proteins histone H2B, RhoA and, HNP‐1. An innovative procedure was applied that makes use of pre‐phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP‐ribosylated peptides, thus showing that the sequence surrounding ADP‐ribosylated residues affects the substrate selectivity of macrodomains.     

Synthesis of sugar nucleotides by application of phosphoramidites

A new method for the construction of pyrophosphates is reported based on the coupling of a sugar phosphate and a nucleoside phosphoramidite. The in situ formed phosphate-phosphite intermediate was subsequently oxidized with tBuOOH. Three UDP-N-acetylglucosamine derivatives were prepared using this one-pot procedure in good yields.     

Rapid fluidic exchange microsystem for recording of fast ion channel kinetics in Xenopus oocytes

We present a new lab-on-a-chip system for electrophysiological measurements on Xenopus oocytes. Xenopus oocytes are widely used host cells in the field of pharmacological studies and drug development. We developed a novel non-invasive technique using immobilized non-devitellinized cells that replaces the traditional "two-electrode voltage-clamp" (TEVC) method. In particular, rapid fluidic exchange was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimetre-sized cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of human epithelial sodium channels (ENaC). Our results show that the response time of this ion channel to a specific reactant is about an order of magnitude faster than could be estimated with the traditional TEVC technique.     

A Cyclic Sulfate Approach to the Synthesis of 1,4-Dideoxy-1,4-imino Derivatives of L-Xylitol,L-Arabinitol and D-Xylitol

Polyhydroxylated pyrrolidines are readily accessible by ring opening of a 1,4-cyclic sulfate function in pentitol derivatives by nitrogen nucleophiles and further processing of the in situ generated charged sulfate group.     

Controlled Microsynthesis of Silica Particles in a Microfluidic Setup

We report on a novel microsynthesis method for easy controlling the size of silica micro- and nanoparticles. Colloids are synthesized in a microfluidic chamber by layer-wise adsorption of silica to precipitated nuclei. We prepare samples of spherical particles with different numbers of coatings and find that particles with less than 15 coating layers are uniform in size. Particles with more coatings are disperse. This finding is attributed to peptization of nanoparticles and independent growth of secondary nanoparticles. For all numbers of layers, the maximum particle size increases with the number of coatings.     

Expression of GD3 synthase modifies ganglioside profile and increases migration of MCF-7 breast cancer cells

B- and c-series of gangliosides are over-expressed in neuro-ectoderm-related cancers, including breast cancer. It has been shown that G_D3 ganglioside is over-expressed in about 50% of invasive ductal breast carcinoma and the G_D3 synthase (GD3S) gene displays higher expression among estrogen receptor (ER) negative breast tumors. We previously showed that GD3S expression in MDA-MB-231 breast cancer cells induces the expression of G_D2 and increased cell proliferation and migration via a G_D2-dependent activation of c-Met receptor. Here, we show that in ER-positive MCF-7 breast cancer cells, GD3S expression resulted in an increase of G_D1b, which was associated with a decrease of G_M1a and G_M2. Meanwhile, GD3S expressing MCF-7 cells exhibited an increased migration without any modification of proliferation rate. Therefore, GD3S expression can result in different modifications of both ganglioside profiles and cell phenotypes depending on breast cell types.     

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.     

Diversity-oriented synthesis of functionalized pyrrolo[3,2-d]pyrimidines with variation of the pyrimidine ring nitrogen substituents

Nine 2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidine-6-carboxylic acid benzyl esters 12 were synthesized in four steps from 4-oxo-N-(PhF)proline benzyl ester 7 by a general method in which elements of molecular diversity were readily added onto the pyrimidine nitrogens. Conversion of 4-oxoproline 7 into the corresponding aminopyrrole 8 using benzyl-, allyl-, and isopropylamine followed by treatment with phenyl, allyl, and ethyl isocyanate gave nine different ureas 9. 4-Ureido-1H-pyrrole-2-carboxylic acid benzyl esters 9 were then converted into the respective pyrrolo[3,2-d]pyrimidines 12 using trichloroacetyl chloride in acetonitrile followed by treatment with Cs(2)CO(3). Crystallization from toluene gave the desired deazapurines in 37-55% overall yield from proline 7.     

Microwave-assisted organic synthesis: scale-up of palladium-catalyzed aminations using single-mode and multi-mode microwave equipment

Batch wise scale-up of Buchwald-Hartwig aminations under microwave irradiation has been investigated for the first time. Multi-mode (microSYNTH and MARS) (several vessels irradiated in parallel per batch) as well as single-mode (Discover) (one vessel irradiated per batch) platforms can be successfully used for this purpose with trifluoromethylbenzene (benzotrifluoride: BTF) as amination solvent. The obtained yields indicate a direct scalability in BTF for all the studied aminations. The Voyager equipment (based on a Discover platform) is the most convenient system since it allows an automatic continuous batch wise production without the necessity to manually load and unload reaction vessels.