Synthesis of thioester peptides for traditional Native Chemical Ligation of the Syk protein and for auxiliary-mediated Native Chemical Ligation

Ruth E Campbell, Purdue University

Abstract

Native Chemical Ligation (NCL) is a highly efficient method of synthesizing large peptides and proteins, such as HIV-1 protease dimer, oxtytocin and human parathyroid hormone (hPTH). NCL is also useful in studying the structure and movement of various proteins, such as the SH2 domain of the Syk protein, an area of research pursued by Dr. Carol Post's laboratory. The Syk protein is a non-receptor protein tyrosine kinase involved in B-cell development, and the structure of its SH2 domains is still unknown. The Post lab studies the SH2 domains by NMR, requiring the domains to be labeled; such labeling can be carried out by NCL of the protein and isotopically labeled synthetic thioester peptides. This thesis thus describes the production of these thioester peptides by Solid Phase Peptide Synthesis (SPPS) followed by coupling of the free C-terminus of the peptide to a thiol nucleophile, forming the thioester. Despite NCL's value, there are some drawbacks to traditional ligation. NCL requires a cysteine residue (one of the rarest natural amino acids) at the site of ligation, and the mechanism of ligation is sensitive to the sterics of the amino acids. These factors limit the range of peptides and proteins that can be ligated via NCL. As such, various auxiliaries have been developed to overcome the difficulties presented by traditional NCL. Photolabile (m)Mhnb auxiliaries (designed and synthesized by Dr. Soo Sung Kang and Dr. Nawaporn (Ning) Sanguantragun) are compounds that permit cysteine-free NCL of sterically bulky peptides. Furthermore, these auxiliaries are photolabile, allowing easy removal with no side-product formation. Using these auxiliaries, the modified NCL mechanism consists of an S to O to N-acyl transfer, following which the native peptide bond is formed with photolytic cleavage of the auxiliary. In order to confirm the proposed mechanism, proof-of-concept studies using a variant of the auxiliary and phenyl thioester are underway in order to prove that the mechanism involves an S to O-acyl transfer. Thioester peptides are required for this proof-of-concept study to demonstrate that the mechanism is the same for various peptide substrates as it is for the phenyl thioester. Herein is reported the synthesis of thioester amino acids and peptides for the auxiliary mediated NCL, using SPPS and coupling reactions of the free C-terminus to the thiol nucleophile.

Degree

M.S.

Advisors

Lipton, Purdue University.

Subject Area

Organic chemistry

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