The study of a prokaryotic glycolytic enzyme

The overall objective of this project is to generate novel carbohydrate binding proteins for use in glycoprotein analysis which are amenable to large scale production. The approach used here is the modification of prokaryotic glycolytic enzymes. Their enzymatic activity will be eliminated while hoping they still retain their binding capabilities. These proteins will be immobilized onto different surfaces to generate advanced bioanalytical platforms which will have huge commercial potential in the field of glycoanalysis

Evaluation of photografted charged sites within polymer monoliths in capillary columns using contactless conductivity detection

Capacitively coupled contactless conductivity detection (C4D) is presented as a novel and versatile means of visualising discrete zones of charged functional groups grafted onto polymer based monoliths. Monoliths were formed within 100 μm UV transparent fused silica capillaries and photografting methods were subsequently used to graft a charged functional monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) onto discrete regions of the “generic” monolith using a photomask. Post-modification monolith evaluation involves scanning the C4D detector along the length of the monolith to obtain a profile of the exact spatial location of grafted charged functionalities with millimetre accuracy. The methodology was extended to the visualisation of several zones of immobilised protein (bovine serum albumin) using photografted azlactone groups to enable covalent attachment of the protein to the monolith at precise locations along its length. In addition, the extent of non-specific binding of protein to the ungrafted regions of the monolith due to hydrophobic interactions could be monitored as an increase in background conductivity of the stationary phase. Finally, the technique was cross-validated using fluorescence microscopy by immobilising green fluorescent protein (GFP) in discrete zones and comparing the profiles obtained using both complementary techniques

Pot, atom and step economic (PASE) synthesis of highly functionalized piperidines: a five-component condensation

The diastereoselective pot, atom and step economic (PASE) synthesis of highly functionalized piperidines has been realized. The procedure simply involves mixing methyl acetoacetate, 2 equiv of aldehyde and 2 equiv of aniline together in the presence of InCl3. In most cases the piperidine precipitates out of solution. (c) 2007 Elsevier Ltd. All rights reserved

Glycolytic enzymes - novel carbohydrate binding proteins for glycoprotein analysis

•The cloning, expression, purification and characterisation of recombinant prokaryotic glycolytic enzymes •The mutagenesis of prokaryotic glycolytic enzymes to generate novel recombinant carbohydrate binding proteins •The characterisation of the binding profile of the novel recombinant carbohydrate binding protein

Combining pot, atom and step economy (PASE) in organic synthesis. Synthesis of tetrahydropyran-4-ones

The combination of pot, atom and step economy (PASE) in the synthesis of organic molecules of medium complexity can lead to a significant 'greening' of a synthetic route. This is demonstrated by the synthesis of highly substituted tetrahydropyran-4-ones and is quantified by a series of recognised metrics, which demonstrate the efficiency of combining PASE over conventional synthetic strategies

Lectin based glycoprotein analysis

Many of the biopharmaceutical therapeutics entering the market and currently in clinical trails are recombinant glycoprotein molecules, the glycan moieties of which have a significant impact on efficacy and immunogenicity. The cell culture techniques required to produce these glycoproteins often result in products that are heterogeneous with respect to glycan content. This inconsistency ultimately leads to increased production costs and restricts patient accessibility to these therapeutics. To overcome these difficulties novel analytical platforms facilitating rapid in-process monitoring and product quality control are essential. Work undertaken within the Centre for Bioanalytical Sciences (CBAS) seeks to exploit the microbial world as a source of novel biorecognition elements to produce such platforms

Production of lectin-affinity matrices for process-scale glycoprotein purification

A selection of prokaryotic lectins with a variety of glycan specificities and affinities have been identified, cloned, expressed in Eschericia coli and characterised. The aims of this project are to: - express the lectins at 1L scale to produce sufficient quantities for immobilisation studies (~100 mg) - immobilisethelectinsonSepharose - evaluate lectin performance on column by monitoring their ability toreproducibly capture and elute glycoprotein glycoforms

Exploiting prokaryotic chitin-binding proteins for glycan recognition

• The cloning, expression and characterisation of prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus luminescens Microfluidics and Photorhabdus asymbiotica • Development of an assay to assess the activity of chitin-binding proteins • Mutagenesis of chitin-binding proteins to alter glycan recognition pattern

An improved synthesis of (2E,4Z)-6-(benzyloxy)-4-bromohexa-2,4-dien-1-ol

An improved synthesis of (2E,4Z)-6-(benzyloxy)-4-bromohexa-2,4-dien-1-ol has been devised. This new route increases the throughput and yield of the diene product by circumventing a low yielding preparation of boronic acid intermediate as well as removing the need to use multi-gram quantities of highly toxic thallium salts. In the process of developing this new route, a higher yielding preparation of ( E)-3-hydroxyprop-1-enylboronic acid was also achieved. (c) 2007 Elsevier Ltd. All rights reserved

Genetically enhanced recombinant lectins for glyco-selective analysis and purification

- Generation of a library of recombinant prokaryotic lectins (RPL’s) through random mutagenesis of the carbohydrate binding sites of bacterial lectins. - Characterisation of mutant lectins with respect to structure and specificity - Provision of mutant RPL’s with enhanced affinity and/or altered specificity, alongside wild-type RPL’s, for glycoprotein analysis and purificatio