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Towards novel luminescent probes for monitoring β- galactosidase activity

Yates, Francesca Jo (2010)
Ph.D. thesis, University of Birmingham.

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This thesis describes efforts made towards the synthesis of a biologically stable, luminescent molecular probe, which could be used to investigate in vivo the processing of sugars by β-galactosidases. To this end, a lactose-based probe was designed, featuring a Lanthanide held within a chelate and appended to the glucosyl unit, and a proximal naphthyl moiety, attached to the galactose unit, which would function as a sensitiser for luminescence. A β-galactosidase enzyme from B. circulans was chosen to carry out the investigation. A number of novel methyl glucosides, functionalised with a naphthyl moiety at C6 of the sugar, were prepared. These were then used as glycosyl acceptors to make disaccharides (lactose analogues), with the enzyme (functioning in reverse) catalysing the glycosylation. The enzymatic reaction was optimised by varying the amount of enzyme, the reaction pH, the ratio of glycosyl acceptor to donor, the reaction temperature, concentration and solvent mixture. The optimal conditions were found to be a 0.4 M reaction solution at pH 7.0 with 20% acetonitrile, a 7:1 ratio of glycosyl acceptor to donor, 19.6 U of enzyme per mmol of acceptor, and a reaction temperature of 30°C. The resulting disaccharide products exhibited unusual regioselectivity for the β-galactosidase from B. circulans, with unexpected β(1→3) and β(1→2) glycosidic linkages being formed. In an effort to increase the efficiency of the process of identifying suitable substrates for the enzyme, a dynamic combinatorial chemistry approach was also explored. This used disulfide bonds to attach the naphthyl moiety to the methyl glucoside using linkers of different lengths. From this library, the enzyme successfully processed the novel disulfide GlcOMe-S-S-CH\(_2\)Np as a glycosyl acceptor with p-nitrophenyl galactose as the glycosyl donor. This resulted in a novel disaccharide featuring a naphthyl group attached via a disulfide bond to the glucosidic residue.

Type of Work:Ph.D. thesis.
Supervisor(s):Cox, Liam
School/Faculty:Colleges (2008 onwards) > College of Engineering & Physical Sciences
Department:School of Chemistry
Subjects:TP Chemical technology
QD Chemistry
Institution:University of Birmingham
ID Code:1376
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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