Andreas Rühmann

Cyclic tetrapeptides via the ring contraction strategy: Chemical techniques useful for their identification

Cyclic tetrapeptides are a class of natural products that have been shown to have broad ranging biological activities and good pharmacokinetic properties. In order to synthesise these highly strained compounds a ring contraction strategy had previously been reported. This strategy was further optimised and a suite of techniques, including the Edman degradation and mass spectrometry/mass spectrometry, were developed to enable characterisation of cyclic tetrapeptide isomers. An NMR solution structure of a cyclic tetrapeptide was also generated. To illustrate the success of this strategy a library of cyclic tetrapeptides was synthesised.

Synthesis of a photoactivatable 9-Z-oleic acid for protein kinase C labeling

Abstract A convenient synthesis of ( Z )-18-[4-[3(trifluoromethyl]phenyl]-9-octadecenoic acid ( 13 ) for photoaffinity labelling is described. Photochemical experiments on 13 and its precursor 11 in ethanol solution as well as in argon matrix 12 K demonstrate the formation of diazo compounds and ultimately carbene-derived products.

Flow cytometric detection of proteolysis in peptide libraries synthesised on optically encoded supports

The concept of optically encoding particles for solid phase organic synthesis has existed in the literature for several years. However, there remains a significant challenge to producing particles that are capable of withstanding harsh solvents and reagents whilst maintaining the integrity and range of the optical encoding. In this study, a new generation of fluorescently encoded support particles was used for both solid phase peptide synthesis and on-particle analysis of proteolysis in a multiplexed, flow cytometric assay. The success of the assay was demonstrated through the use of a model protease, trypsin. Our results show that the use of solid supports with high peptide yield, high swellability in water and high penetration of the enzyme into the interior of the particle is not absolutely necessary for proteolysis assays.

Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

Bead‐based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two‐sided t‐tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5‐labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 × 104 to 500 × 104 molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead‐based cytometry assays to ensure reproducibility in inter‐ and intra‐platform comparisons of data between laboratories.