Development of New Synthetic and Analytical Tools in Glycobiotechnology
Carbohydrates (such as sugars, glycans and polysachharides) provide the largest biomass on Earth and are central to many aspects of biotechnology. As such glycoscience and glycobiotechnology have broad applications in the development of biofuels, biomaterials and food stuffs from recycled and readily available biomass, as well as in medical research. Through investigation of the chemistry, structure and interactions associated with various carbohydrate molecules, it is possible to understand mechanisms of disease, develop novel glyco-based therapeutics and replicate the natural biosynthesis of glycans for synthetic purposes. However, the complex and diverse molecular nature of carbohydrates means that there is an urgent need to develop robust, wide-ranging synthetic and analytical methodologies for general use in this area.
In the Flitsch lab. we have developed, and are continuing to expand, a toolbox for the synthesis and analysis of complex carbohydrates in order to understand their function. The main focusses of this research are:
(i) glycoarrays to study glycoenzyme activity and discover carbohydrate-protein interactions
(ii) ion mobility mass spectrometry for high resolution structural analysis of carbohydrates
(iii) mass spectrometry for the label free identification of carbohydrate-binding proteins
(iv) chemoenzymatic synthesis of glycoconjugates (glycopeptides, glycolipids, etc.)
Gray C. J; Thomas B, Upton R, Migas L. G, Eyers C. E, Barran P. E and Flitsch S. L. “Applications of ion mobility mass spectrometry for high throughput, high resolution glycan analysis.” BIOCHIMICA AT BIOPHYSICA ACTA (BBA) – GENERAL SUBJECTS. DOI: 10.1016/J.BBAGEN.2016.02.003 – FEB 6 2016.
Both, P.; Green, A. P.; Gray, C. J.; Sardzik, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.; Richardson, D.; Field, R. A.; Widmalm, G.; Flitsch, S. L.; Eyers, C. E. Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing. NATURE CHEMISTRY. 6(1); 65 – 74. DOI 10.1038/NCHEM.1817 – JAN 2014
Sardzik, Robert; Green, Anthony P.; Laurent, Nicolas; Both, Peter; Fontana, Carolina; Voglmeir, Josef; Weissenborn, Martin J.; Haddoub, Rose; Grassi, Paola; Haslam, Stuart M.; Widmalm, Goran; Flitsch, Sabine L. Chemoenzymatic Synthesis of O-Mannosylpeptides in Solution and on Solid Phase. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 134(10); 4521 – 4524. DOI 10.1021/ja211861m – MAR 14 2012
Castangia, Roberto; Austeri, Martina; Flitsch, Sabine L. Enzymatic Amine Acyl Exchange in Peptides on Gold Surfaces. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 51(52); 13016 – 13018. DOI 10.1002/anie.201205404 – 2012
Enzymatic Cascades in Biocatalysis
The use of catalysis in modern chemical methods allows reactions to be run quickly and efficiently with lower energy input than uncatalysed procedures. Whilst many man-made catalysts are difficult to synthesise and often incorporate expensive, toxic and / or finite resources, enzymes offer a natural, more renewable alternative. A great advantage of these biocatalysts is that their lowering of energy input allows reactions to be run under ambient conditions and often in a highly selective manner. Another benefit of biocatalysis is that, in Nature, cascades of enzyme-catalysed reactions occur together in pathways allowing tandem catalysis to perform complex, multistep chemical transformations in a single cell.
Through the use of modern molecular biology techniques, it is possible to transfer individual enzymes from their host organism and produce them in more amenable laboratory strain bacteria. This allows them to be more easily studied, engineered and integrated into new enzyme cascades, both in whole cell systems and as cell-free formulations. In the Flitsch group we are interested in the tailoring of various enzymes to perform specific, synthetically-useful reactions and incorporating these into bespoke pathways for the production of industrially- and biomedically-relevant compounds. Examples of target products of particular interest include high value chiral amines as pharmaceutical / fine chemical precursors and glycoconjugates as chemical biology probes for glycobiotechnology.
P. Both, H. Busch, P. P. Kelly, F. G. Mutti, N. J. Turner, S. L. Flitsch, Sabine. Whole-Cell Biocatalysts for Stereoselective C-H Amination Reactions. J. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION., 55; 1511 – 1513.DOI: 10.1002/anie.201510028– JAN 22 2016
Noble, Gavin T.; Craven, Faye L.; Voglmeir, Josef; Sardzik, Robert; Flitsch, Sabine L.; Webb, Simon J. Accelerated Enzymatic Galactosylation of N-Acetylglucosaminolipids in Lipid Microdomains. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 134(31); 13010 – 13017. DOI 10.1021/ja302506t – AUG 8 2012
Rannes, Julie B.; Ioannou, Avgousta; Willies, Simon C.; Grogan, Gideon; Behrens, Carsten; Flitsch, Sabine L.; Turner, Nicholas J. Glycoprotein Labeling Using Engineered Variants of Galactose Oxidase Obtained by Directed Evolution. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 133(22); 8436 – 8439. DOI 10.1021/ja2018477 – JUN 8 2011