טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLaps Shay
SubjectPalladium Assisted Synthesis and Manipulation of Peptides
and Proteins
DepartmentDepartment of Chemistry
Supervisor Professor Ashraf Brik
Full Thesis textFull thesis text - English Version


Abstract

The great technological innovations that enabled the preparation of proteins by molecular biology approaches, opened for the scientific community new exciting opportunities to investigate and reveal the mysteries behind the fascinating biochemical processes that proteins are involved in. Proteins are the building blocks of life and are crucial for the catalysis of vital biochemical reactions, muscles building, delivery of oxygen molecules in the blood and more. In particular, proteins play dominant role in the development of diseases such as Cancer, Alzheimer, etc. Understanding of their exact role and mechanism of actions as well as their structural properties may bear a huge potential to discover and develop new lifesaving drugs and vaccines. Nevertheless, complex proteins with posttranslational modifications (PTM's) are often difficult to study using molecular biology approach. Chemical synthesis of proteins has opened the opportunity to investigate these complex proteins sine it enable their preparation in high efficiency, high purity and in large scale. In order to chemically synthesize complex proteins with PTM's at desired sites and/or interesting functional groups, there is a need for orthogonal chemistry that can be carried out at mild physiological conditions, on different and selected sites in peptides or proteins and enable controlled manipulations. The uses of different transition metals (e.g. the toxic mercury) for the deprotection of cysteine (Cys) protecting groups (PG's) and the wide use of palladium for the modification of proteins in aqueous media, triggered us to investigate the potential of palladium to assist in the chemical synthesis of complex peptides and proteins under mild conditions that can be useful for future research in living biological systems. In our research, we designed and developed a thiazolidine chemical linkage within the backbone of protein which can be cleaved in the presence of Pd (II) complexes, to give two parts of the protein that contains different functional groups under mild conditions. In addition, we discovered a new and efficient Cys(t-butyl) unmasking reaction under physiological conditions based on palladium chemistry. Moreover, we discovered and developed interesting and useful system of three different Cys PG's that can be removed in orthogonal manner using Pd (II) complexes depending in the used reaction conditions. By utilizing these findings, we developed a novel method for the preparation of library of peptides via orthogonal deprotection and selective modifications at three different sites. Moreover, we succeeded used these conditions to chemically synthesize for the first time the Copper Storage Protein-1 that was very difficult to prepare without the innovative chemical methods we have introduced in this study.