טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentGuy Mann
SubjectDevelopment of Palladium Based Cleavable Linkers for
Peptide and PROTEIN Chemistry
DepartmentDepartment of Chemistry
Supervisor Full Professor Brik Ashraf
Full Thesis textFull thesis text - English Version


Abstract

Many multi-functional materials are based on covalently linking complex molecules to one another, and then separating them at a desired time. As such, the design and development of cleavable linkers is imperative for many research applications. Cleavable linkers have long been applied in many fields of organic synthesis, and their usage has even expanded to include in vivo applications and drug development. This versatility is mainly due to the diversity of linkers and cleavage trigger reactions.

Cleavable linkers are imperative for the synthesis of peptides and proteins. Solid phase peptide synthesis (SPPS) is a procedure in which a peptide’s carboxylic terminal (C-terminal) is linked to a solid support, thus allowing for multiple chemical reactions to be conducted without cumbersome purification steps. SPPS has revolutionized total chemical synthesis of peptides and proteins, and has allowed for the direct production of unprotected peptides and proteins at lengths ranging from 30 to 50 mer. Synthetic peptides can include a uniform array of complex modifications that deviate from the 20 amino acids naturally available for recombinant expression. This flexibility then allows for the synthesis of uniformly-modified complex targets with high purity.

The scope of SPPS can be further expanded by combining SPPS with chemo-selective ligation approaches such as native chemical ligation (NCL). In this approach, the peptide or protein sequence is split into peptide fragments, and then undergoes chemo-selective ligation in an aqueous solution. The combination of SPPS and NCL paved the way for the synthesis of many complex protein targets, but it also has some limitations. One limitation, for example, is that the uneven distribution of hydrophobic amino acids in the peptide sequence leads to issues of solubility for SPPS-generated peptide fragments. This issue can be combated through the introduction of reversible solubilizing tags which increase the solubility of the peptide intermediates, thus increasing the scope of SPPS.

In this work, we describe the design of a new cleavable linker “aminophacm” based on the existing Cys protecting group phenylacetamidomethyl. We then explore the "one pot" application for hydrophobic peptide and protein syntheses, followed by palladium complex-induced cleavage in aqueous conditions to allow for the removal of the solubility tag upon completion of the ligation steps. Palladium was chosen for the tag cleavage due to its high affinity to the thiol in Cys, allowing for high efficiency and specificity. We then applied our method for the efficient preparation of histone 4 (H4) protein by attaching a temporal solubility tag to Cys side chains during SPPS, followed by one-pot ligation, solubilizing tag removal and desulfurization.