This thesis is composed of two separate projects: Kinetic Template-Guided Tethering of Fragments and Design and
Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1 Degradation after
DNA Damage.
Kinetic Template-Guided Tethering of Fragments
The development of a novel methodology for the site-directed discovery of small molecule, protein-binding ligands is
described. The protein of interest, with a cysteine thiol (either native or engineered) adjacent to the desired binding
pocket, is incubated with mixtures of low molecular weight compounds (fragments) modified with either an
acrylamide or a vinyl sulfonamide capture group. Any ligand within the mixture that binds within the pocket brings
the capture group into close proximity with the cysteine thiol, promoting a conjugate addition reaction at an increased
rate over the background reaction. The capture reaction is designed to be slow, such that during the time course of an
experiment, no adduct formation is observed unless the reaction is templated by the protein. By this method, binding
ligands are rapidly identified by mass spectrometry analysis of the crude reaction mixtures. The methodology has
been termed ‘kinetic template-guided tethering’. Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1
Degradation after DNA Damage
An inhibitor described within the literature was found to attenuate the reduction of cyclin D1 after DNA damage to
cells. In order to implement a two-step chemical proteomics strategy to find the molecular target of this inhibitor, a
synthesis of the compound with an alkyne appendage was required. The alkyne acts as a functional handle for
attachment of a reporter molecule in cells via the Huisgen cycloaddition (‘Click’) reaction. The design and synthesis
of this inhibitor with the alkyne appendage is described.