Principal Supervisor: Professor Martin Wills, Department of Chemistry
Co-supervisor: Dr Christophe Corre, Department of Chemistry / School of Life Sciences, Professor Peter O’Connor, Department of Chemistry
PhD project title: A New Bioconjugation Reagent for the Biosciences.
University of Registration: University of Warwick.
a) Background. There is a need for efficient chemical reactions which permit the attachment of chemical groups such as dyes, fluorescent markers, drug molecules etc., to large biomolecules such as proteins or DNA. Transformations of this type, i.e. ‘bioconjugation’ reactions,1 are used for both the identification and treatment of a range of diseases and medical conditions. A fluorescent group attached to a targeting protein, for example, can help identify the location of the early development or ongoing progression of a specific illness such as a cancer. The attachment of a small-molecule drug to an antibody with specificity for cancer cell antigens provides a tool for the development of antibody-drug conjugate medicines (ADCs).2 However, efficient bioconjugations are generally difficult– multiple sites may react and undesired side-reactions may occur. For this reason, efficient reactions, which do not interfere with normal biological processes (i.e. ‘bioorthogonal’ reactions), have been developed. An important ‘bioorthogonal’ reaction is the cycloaddition of a ‘strained’ alkyne with functionalised azides without the need for a copper catalyst (Figure 1).3
b) Programme of work. In recent work at Warwick, recently published,4 a new class of strained alkyne reagent has been developed and a number of derivatives are now available for assessment as bioconjugation reagents (Figure 1). This project shall involve a study of its attachment to a range of proteins, together with analysis of the properties of the resulting products. Analysis of the products shall be undertaken using a range of mass spectrometry methods, including FTICR and MALDI; which are well suited to protein analysis. In addition, high field solution NMR and circular dichroism (CD) methods will be employed in order to determine the effect of the bioconjugation on the protein structure. The reaction of the bioconjugated proteins with azide-containing dyes in a single step will lead to the formation of fluorescent compounds which will be analysed by UV spectroscopy. The rates of addition of the strained alkynes to azides will be measured, in order to gain structure-reactivity information. Overall a rapid picture will be established of the effectiveness of the new bioconjugation agents on example proteins using a range of analytical methods, and of the potential value of the methodology for the development of diagnostic agents with medical applications.
Several proteins will be closely related to a series which have been identified in ongoing studies of protein-protein and protein-DNA interactions within the Departments of Chemistry and Life Sciences. This interdisciplinary project would be ideal for a Biochemistry or Chemistry graduate student with an interest in working at the boundary between these two areas. The student would gain training in both synthetic chemistry and molecular biology, as well as a range of analytical techniques in both disciplines, and would be well qualified to work in this area in an academic or industrial research environment. There is a also project in parallel with this one in collaboration with AstraZeneca, funding a PhD studentship (filled) for four years.
- Reviews on click chemistry and applications: (a) P. Thirumurugan, D. Matosiuk and K. Jowiak, Chem. Rev. 2013, 113, 4905-4979. (b) J.M. Palomo, Org. Biomol. Chem. 2012, 10, 9309-9318.
- Click reactions in peptide-based drug design; J. D. Thomas, H. Cui, P. J. North, T. Hofer, C. Rader, and T. R. Burke Jr. Bioconjugate Chem. 2012, 23, 2007–2013.
- Cu-free click cycloadditions in chemical biology; (a) J.C. Jewett and C. R. Bertozzi, Chem. Soc. Rev. 2010, 39, 1272-1279. (b) X. Zhang and Y. Zhang, Molecules 2013, 18, 7145-7159. (c) Y. Gong and L. Pan, Tetrahedron Lett. 2015, 56, 2123-2132. (d) X. Chen, K. Muthoosamy, A. Pfisterer, B. Neumann and T. Weil, Bioconjugate Chem. 2012, 23, 500-508. (e) J. Dommerholt, F. P. J. T. Rutjes and F. L. van Delft, Top. Curr. Chem (Z) 2016, 374:16.
- Our preliminary publication on these results; Strained alkynes derived from 2,2′-dihydroxy-1,1′-biaryls; synthesis and copper-free cycloaddition with azides, A. Del Grosso, L.-D. Galanopoulos, C. K. C. Chiu, G. J. Clarkson, P. B. O′ Connor and M. Wills, Org. Biomol.Chem. 2017, 15, 4517 – 4521.
BBSRC Strategic Research Priority: Molecules, cells and systems
Probably most closely aligned to: Technology Development for the Biosciences.
In terms of MIBTP, the area most closely aligned would be the Molecular Mechanisms part of Molecules, Cells and Systems, since the methodology will be employed to follow biological mechanisms. However the project would also be related to some extent to Bioenergy and Industrial Biotechnology as well.
Techniques that will be undertaken during the project:
- Synthetic organic chemistry; Synthesis of substrates for testing, detection, isolation and analysis of enzyme reaction products, use of analytical methods including Nuclear Magnetic Resonance, Mass Spectrometry (including MALDI), and chromatographic methods. Supervisor; Professor Martin Wills.
- Molecular biology; including use of restriction enzymes, cloning, PCR, characterisation of proteins and protein/protein and protein/DNA interactions through kinetic studies using methods such as UV spectrometry and circular dichroism. Supervisor; Dr Christophe Corre.
- State of the Art Mass Spectrometry; including complex MS/MS analyses with FTICR and MALDI instruments and comparisons with protein-protein interaction databases to interpret protein/protein interactions. Supervisor Prof Peter O’Connor.
In all cases, the respective laboratories are equipped to a high standard with the facilities and instruments to support this project.
Contact: Professor Martin Wills, University of Warwick
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