An integrated on-chip sirtuin assay

A microchip-based assay to monitor the conversion of peptide substrates by human recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused silica microchip consisting of a microfluidic separation structure with an integrated serpentine micromixer has been used. As substrate for the assay, we used a 9-fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known substrate of hSIRT1. The Fmoc group at the N-terminus resulting from solid-phase peptide synthesis enabled deep UV laser-induced fluorescence detection with excitation at 266 nm. The enzymatic reaction of 0.1 U/μL hSIRT1 was carried out within the serpentine micromixer using a 400 μM solution of the peptide in buffer. In order to reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose. The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The approach was successfully utilized to screen various SIRT inhibitors.     

(Pentaphenyl)aluminacyclopentadiene as a complexing ligand. The molecular structure of (pentaphenyl)aluminacyclopentadiene and its complex with 1,5-cyclooctadienenickel

The molecular conformations of a (pentaphenyl)aluminacyclopentadiene ligand (I) and its complex with 1,5-cycloactadienenickel (II) have been determined from single crystal X-ray data collected at room temperature with counter methods. The free ligand crystallizes in the monoclinic space group Cc with 4 molecules in a unit cell of dimensions a 10.5598(5), b 22.7089(12), c 13.3417(4) Å, β 98.064(3)°; its (COD)Ni complex Crystallizes in the monoclinic space group P2₁/n with 4 molecules in a unit cell of the dimensions a 11.9948(8), b 16.9758 (13), c 18.7721(14) Å. β 97.958(3)°. Both structures have been refined anisotropically to R values of 0.0577 and 0.0493, respectively. Upon complexation of I to the nickel atom the planarity of the ring system is distorted, with the aluminum being bent away from the nickel. A direct metal—metal interaction in II (NiAl: 2.748(1) Å) cannot be ruled out.     

Monitoring on-chip Pictet-Spengler reactions by integrated analytical separation and label-free time-resolved fluorescence

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.     

Precision-cut liver slices from rats of different ages: basal cytochrome P450-dependent monooxygenase activities and inducibility

The biotransformation capacity – of the cytochrome P450 (CYP) system for example – is lower but inducibility is more pronounced in neonates than in adults. On the other hand, both enzyme activities and inducibility decline with senescence. Precision-cut rat liver slices are widely used as an in vitro tool for the examination of drug toxicity, xenobiotic metabolism or enzyme induction. The aim of the present study was to assess whether age-related changes in CYP activities and induction observed in vivo are also mirrored in vitro in liver slices. For this purpose, different CYP model reactions were measured in precision-cut liver slices from one-day-old, 40-day-old and one-year-old rats after in vitro exposure to various inducers. Similar to the in vivo situation, basal CYP activities were distinctly lower and inducibility was much more pronounced in liver slices from neonatal than in those from adult animals. Also, enzyme activities were mostly somewhat lower in liver slices from aged rats compared to those from 40-day-old rats. However, CYP inducibility was less pronounced than with younger animals too. Thus, precision-cut rat liver slices are a suitable in vitro tool for investigating age-related changes in CYP activities and induction as well as developmental differences in drug metabolism and toxicity.