Advances in organometallic and protein chemistry.
Doctoral thesis, UCL (University College London).
This thesis describes two areas of scientific investigation. The first contains a description of a study on the synthesis of biotinylated and fluoresceinylated bromomaleimide based reagents. Upon synthesis, the ability of these reagents to add reversibly to cysteine containing proteins is investigated by a series of LCMS experiments. A single point mutant (L111C) of the SH2 domain of the Grb2 adaptor protein, containing a single cysteine residue, is chosen as an ideal protein for study. Thus biotinylated and fluoresceinylated mono and dibromomaleimide reagents are added to Grb2 at 0°C, 21 °C and 37 °C for 2 h at pH 7 and 8 to give protected Grb2-bromomaleimide adducts in high yield and removed using 100 eq of beta-mecaptoethanol for 4 h to return original Grb2 protein intact. Reversibility is shown to be abolished by hydrolysis of the maleimide motif, a process more prevalent in the fluoresceinylated and dibrominated maleimide reagents than for the biotinylated and monobrominated reagents. LCMS is used to investigate the insertion of these reagents reversibly into the single disulfide bridge of somatostatin, a process shown to be complete within 1 h at 21 °C and pH 6. Reduction using 100 eq of beta-mecaptoethanol is shown to take place within 1 h. Additionally no hydrolysis is observed at pH 6, suggesting that with careful control of pH, reversibility of the bromomaleimide reagents can be switched on and off. The second part of the thesis contains a study on mechanistic aspects of organopalladium catalysis, particularly the factors affecting the final reductive elimination stage of the palladium catalysed alkyl amination reaction. DFT calculations have been used to obtain a number of energy level diagrams for the potential energy surface of Pd(IBu)(neopentyl)(morpholide), the three coordinate palladium complex believed to be the species from which reductive elimination takes place. From this it has been found that two different pathways are available; reductive elimination or morpholide promoted C-H activation of the neopentyl motif, the latter of which was favoured. The reaction pathway for reductive elimination from Pd(IBu)(neopentyl)(morpholide) is compared with Pd(IBu)(phenyl)(morpholide), Pd(PCyp3)(neopentyl)(morpholide) and Pd(IBu)(methyl)(morpholide), to show that the phenyl system is considerably lower in energy. The energy requirements for (i) reductive elimination, (ii) morpholide promoted C-H activation and (iii) β-hydride elimination from Pd(IBu)(2-dimethylpropyl)(morpholide) are compared, and shown to be in the order of energy (i)>(ii)>(iii). Additionally, the rate of reaction increases as the number of available reaction sites increases, again making reductive elimination the least favoured process. Offline ESI(+)-MS analysis have been used to monitor the reaction progress of Pd(IBu)(neopentyl)(morpholide) and Pd(IBu)(phenyl)(morpholide) three coordinate complexes. Whilst transient palladium-bound species can be observed for Pd(IBu)(phenyl)(morpholide), for Pd(IBu)(neopentyl)(morpholide) this is not the case, an artefact of the superiority of the sp2 phenyl system over the sp3 alkyl system.
|Title:||Advances in organometallic and protein chemistry|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of BEAMS > Faculty of Maths and Physical Sciences > Chemistry|
Archive Staff Only