Treatment of inflammatory bowel disease by chemokine receptor-targeted leukapheresis.

Leukapheresis removes circulating leukocytes en route to the target organ. Hitherto unspecific matrixes have been used to remove leukocytes in inflammatory bowel disease (IBD). This report describes a novel selective leukapheresis column based on chemokine-chemokine receptor interaction.

Mixed SAMs and MALDI-ToF MS: preparation of N-glycosylamine derivative and thioctic acid methyl ester bearing 1,2-dithiolane groups and detection of enzymatic reaction on Au.

Herein, we report an enzymatic galactosylation reaction of β-glucopyranosylamide 4 and thioctic acid methyl ester 5 bearing 1,2-dithiolane groups to form a new system of mixed self-assembled monolayers (SAMs) on gold. Characterization of the enzymatic activity was conveniently achieved by mass spectrometry.

9-Aminoacridine peptide derivatives as versatile reporter systems for use in fluorescence lifetime assays.

A novel long lifetime fluorescence reporter based on 9-aminoacridine was designed, the lifetime of which can be modulated in a defined manner when in proximity to a tryptophan residue enabling fluorescence lifetime based biochemical assays to be configured.

A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening.

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide.

Real-Time Imaging of Protease Action on Substrates Covalently Immobilised to Polymer Supports.

We report for the first time single bead spatially resolved activity measurements of solid-phase biocatalytic systems followed in real-time. Trypsin cleavage of Bz-Arg-OH and subtilisin cleavage of Z-Gly-Gly-Leu-OH each liberate a free amino group on aminocoumarin covalently immobilised to PEGA(1900) beads [a co-polymer of poly(ethylene glycol) with molecular mass of 1900 cross-linked with acrylamide]. This restores fluorescence which is imaged in optical sections by two-photon microscopy.

Controlling protein retention on enzyme-responsive surfaces.

The ability to change the properties of solid surfaces on demand is a key component of a multitude of established and emerging technologies. Stimuli that have previously been used to trigger changes in surface properties include changes in solvent, light, pH, ionic strength, temperature and magnetic or electric fields. We are interested in developing surfaces that can be triggered by the catalytic action of enzymes.

Enzyme-cleavable linkers for peptide and glycopeptide synthesis.

Hydroxymethylphenoxy linkers that are commonly used in solid phase peptide synthesis are surprisingly susceptible to efficient cleavage by the protease chymotrypsin with a broad range of amino acid residues being tolerated at the scissile bond; this enzyme-cleavable linker system has been applied to peptide and glycopeptide synthesis.

Optimized polymer-enzyme electrostatic interactions significantly improve penicillin G amidase efficiency in charged PEGA polymers.

Hydrolytic yields as high as 80% were obtained by using penicillin G amidase (PGA) on substrates anchored on optimized positively charged PEGA polymers. By increasing the amount of permanent charges inside the polymer, electrostatic interactions between the positively charged PEGA(+) and the negatively charged PGA (pI = 5.2-5.

Synthesis of N-linked glycopeptides on solid support and their evaluation as protease substrates.

A range of glycopeptides containing protease cleavage sites were synthesized on solid support using Fmoc-based solid phase glycopeptide synthesis. The immobilized peptides were studied as substrates for the proteases chymotrypsin and thermolysin. For chymotrypsin, N-glycosylation of an Asn residue at the P(2) site appears to reduce hydrolysis whereas glycosylation of the P(1) site does not appear to affect peptide hydrolysis by thermolysin.

Synthesis and modifications of carbohydrates, using biotransformations.

Enzymes continue to be used as important catalysts, for the generation of rare and 'unnatural' monosaccharides and for the selective formation of glycosidic linkages. Multi-enzyme systems have been employed in one-pot strategies for multistep reaction sequences and for co-factor regeneration. The efficiency of glycosidases for glycosylation reactions has been dramatically increased by active-site mutagenesis to generate glycosynthases.