Date of Award
Chemistry and Biochemistry
Gregory R. Wiedman, Ph.D.
Monika Raj, Ph.D.
Joseph Badillo, Ph.D.
petasis, secondary, modification, amine, specific, bioconjugation
Chemical protein synthesis is an invaluable tool that enables the construction of novel protein design, incorporation of non-native functionalities, elucidation of structure-function relationships and enable a wide diversity of modifications (such as Post Translation Modification) to study protein functions. Native Chemical Ligation (NCL) in conjunction with Fmoc Solid Phase Peptide Synthesis (SPPS) is the most used method for synthesis of functional peptides and proteins. The synthesis relies on the reaction of a C-terminal peptide thioester and a N-terminal cysteine peptide. Fmoc SPPS of peptide thioester for chemical protein synthesis via NCL is a challenge. Methods that exist for the synthesis of peptide thioester by Fmoc SPPS require special resins, linkers, additional chemistry and is difficult and time consuming. Therefore, a simple and robust method to synthesize peptide thioester is highly desirable. Chapter two of this thesis describes the development of a versatile approach for direct synthesis of peptide thioesters from a solid support utilizing Fmoc chemistry. The method utilizes a cyclic urethane activation technique for the synthesis of peptide thioesters directly from solid support. The resulting thioester is stable and free of epimerization. The usefulness of this methodology was demonstrated by the synthesis of a 19 amino acid peptide thioester, which was utilized for the synthesis of a 29 amino acid long peptide derived from rabies virus glycoprotein (Rvg) using NCL. This accessible and robust Fmoc-based thioesterication technique provides a significant advance to chemical protein synthesis due to its uncomplicated nature, whereby eliminating special precautions and additional steps typically needed for synthesis of peptide thioesters. This approach can be used to incorporate post-translational modifications for the synthesis of complex post translational peptides and proteins in milligram quantities that can be used to study their structure and function. This modification approach can also be used for the synthesis of other complex protein modifications through residue specific N-terminal chemical modification by NCL. Chemical modification of protein is a critically important tool for various biological applications such as probing protein dynamics, elucidating protein structure and functions, enhancing protein stability in biological system and construction of protein-drug conjugates. Traditional modification techniques utilize non-specific lysine and cysteine conjugation strategies that produces a heterogenous mixture of conjugates. Over the last two decades there has been an increasing need to developed methods which would modify protein in a controlled manner. Chapter three describes the development of a novel site-specific Secondary Amine Selective Petasis (SASP) bioconjugation strategy using Petasis reaction to modify secondary amines and N-terminal proline. The SASP reaction has been shown to modify a wide variety of peptides and proteins with high selectivity for N-terminal proline. The resulting bioconjugate is highly stereoselective (de >99%) which can be useful in drug discovery. Also, the multicomponent nature of this conjugation technique enables dual labeling of complex proteins in one pot with various cargoes such as dye, biotin and alkynes. The applicability of the SASP bioconjugation technique was demonstrated on a variety of different peptides and proteins with various aldehyde and organoboronate derivatives. The chemo-, regio- and site-specific nature of this method will enable the construction of biomolecular hybrids that can be used to study protein functions, identify new drug targets, deliver potent therapeutics to cellular targets, and engineer new materials. This strategy also provides a powerful tool to study the function and dynamics of post-translational modification such as mono methylated lysine that regulates transcription factor function.
Sim, Yonnette, "Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins" (2020). Seton Hall University Dissertations and Theses (ETDs). 2776.