Development of artificial metalloenzymes via covalent modification of proteins (original) (raw)

II.4.7 Chemical modification of proteins a. Conjugation with carboxylic acid functionalized phosphanes 1-4 b. Covalent modification with N-(4-(diphenylphosphane sulfide)benzyl) maleimide c. Coupling of maleimide functionalized azide and alkynes 5-7 d. Attempts to introduce BH3 protected phosphanes into alkyne and azide functionalized proteins via click-reaction e. Covalent modification with maleimide functionalized phosphaneboranes 10-12 f. Procedures tested for deprotection of phosphane-boranes coupled to proteins g. Coupling of phosphane ligands via hydrazone linkage III. Site-selective bioconjugation of nitrogen-containing ligands to structurally diverse protein hosts Firstly, I would like to thank my PhD supervisor, Prof. Paul Kamer for entrusting me with this interesting and challenging project, for his encouragement, help and academic supervision. I am also grateful to Prof. Ron Wever and Dr Paul Wright for agreeing to be the coexaminers in my dissertation committee. Special thanks to Dr Wouter Laan for his guidance during these years, for being a very demanding, rigorous and constructive scientific supervisor and for kindly proofreading the thesis. I would like to thank Prof Garry Taylor for allowing the use of laboratory space and equipment at the Centre for Biomolecular Sciences-St Andrews. I would like to acknowledge the BSRC Mass Spectrometry and Proteomics Department team, in particular Dr Catherine Botting and Dr Sally Shirran for helpful discussions and technical support in performing the LC-MS measurements and also Mr. Alex Houston for the MALDI TOF measurements. I would also like to thank the Solution-phase NMR Spectroscopy Service team, Dr Tomas Lebl and Mrs Melanja Smith for their help for the NMR measurements. I am thankful to Prof. Malcolm White for allowing us to perform the fluorescence measurements in his laboratory. I acknowledge the EPSRC National Mass Spectrometry Service Centre-Swansea for the accurate mass measurements performed there. I thank Prof. David A. Bernlohr (University of Minnesota) for kindly providing the pRSET-ALBP plasmid, as well as Dr Julio J. Caramelo (Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires), for kindly providing the pET22b(+) Δ98Δ IFAB plasmid. This work was financially supported by European Union Marie Curie Excellence Grants (MEXT-2004-014320). I am very thankful to all former and current colleagues from the Hom-Cat group. I have to start with thanks for our special colleague Bert, for his joyfulness and v brightness. Special thanks for Serghei, Bianca, Gregorio, Peter, Arnald, Debbie and Jason for their fruitful discussions and help with the organic chemistry project but also for being great friends and exceptional lab mates. I am indebted also to Yanmei and Nikos for the synthetic chemistry part. I want to thank Christine, Tanja and Michiel for the good and efficient time we spent together as a team. I am especially grateful to the Applecross girls for the breath of fresh air… provided every day when it was crucially needed. I feel very lucky and fulfilled for having the chance to meet interesting people and to make great friends here in the Chemistry Department, and I must thank Jenny, Bianca, Marzia, Aga, Jacorien and Louise for their permanent encouragements, support and friendship. Special thanks also for Elena, Alma, Cristian, Dragos and Andrei, for bringing into St Andrews the flavour of home. Many thanks also to Prof. Mircea Bulancea and Prof. Rodica Segal for their trust and encouragements at the beginning of my career. Without their initial support I would not be here today. Most importantly, I am grateful to my very special and loving family, for their permanent support. Special thanks to my sister for finding the strength to encourage and help me whilst she has to deal with her own life threatening problems, for my brother for being such a helpful, reliable and honest person and to my parents for teaching me through their personal examples the real values of life. Particular thanks to my daughter, who bravely started a new life in harsh conditions. I have to thank you for being so mature and determined in your work and for being such a good, patient and understanding person during my worst and best days here. What does not kill you makes you stronger and we are both alive! Last but not least, I want to thank God for giving me a second chance in life! vi