Amide‐Forming Ligation Reactions

Abstract

One of the long‐standing pursuits of synthetic organic chemists is the development of chemical methods for the synthesis of peptides and proteins as tools for understanding biological processes and providing therapeutic solutions to human diseases. The advent of chemoselective ligation reactions for the chemical synthesis of peptides and proteins has enabled synthetic chemists to realize this long‐held dream. In an amide‐forming ligation reaction, two uniquely placed functional groups within the peptide allow peptide or protein segments to be joined in a chemoselective manner with an amide bond. In this chapter, some of the early methods based on principles of the capture/rearrangement strategy for ligation of peptides are catalogued, followed by some of the most versatile and well‐established contemporary methods for the preparation of linear/cyclic peptides and proteins such as the native chemical ligation (NCL) and the α‐ketoacid–hydroxylamine (KAHA) ligation. The chapter concludes with some of the most promising new generation ligation methods such as the potassium acyltrifluoroborate–hydroxylamine (KAT) ligation. The tremendous progress in the past two decades in the development of several ligation methods that rely on a pair of unique functional groups is set to expand the horizons of fully functional proteins and multi‐protein assemblies that are purely synthetically prepared. Although contemporary ligation reactions do not match the speed and efficiency at which proteins are assembled in biological systems, the repertoire of chemoselective amide‐forming ligation methods has already enabled synthetic protein chemistry to surpass biology in terms of chemical and structural diversity.

Förster Resonance Energy Transfer Assay for Investigating the Reactivity of Thioesters in Biochemistry and Native Chemical Ligation

“…Often, an aryl thiol “catalyst”, such as MPAA, is added to accelerate the trans-thioesterification step and to prevent non-productive thioester adducts on internal cysteine residues . The thiol catalyst also maintains a reducing environment; however, TCEP is often applied as an additional reducing reagent at concentrations ranging from 10 to 200 mM, although protocols without the addition of TCEP have also been reported . Lastly, aryl thiol additives prevent the unwanted desulfurization of the cysteine side chain caused by TCEP .…”

Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes

“…Remarkable advances in chemical protein synthesis provide access to small- and medium-sized synthetic proteins and enable the site-specific incorporation of post-translational modifications or the preparation of mirror image molecules, including the synthesis of a racemic mixture of Rv1738 to facile protein crystallization by Kent, Baker, and co-workers and the synthesis of d -amino acid polymerase by Liu, Zhu, and co-workers . Other notable achievementschosen from a large number of impressive reportsinclude Muir’s and other group’s preparation of modified histones, , Payne’s sulfated proteins with antithrombotic and anticoagulant activities, and the synthesis of homogeneous polyubiquitin chains by Brik and co-workers and Liu and co-workers. , …”

“…Remarkable advances in chemical protein synthesis provide access to small and medium sized synthetic proteins and enable the site-specific incorporation of posttranslational modifications or the preparation of mirror image molecules, including Kent's and Baker's synthesis of racemic mixture of Rv1738 to facile protein crystallization 22 and Zhu's and Liu's synthesis of Damino acid polymerase. 23 Other notable achievements -chosen from a large number of impressive reports 24 include Muir's and other group's preparation of modified histones, 25,26 Payne's sulfated proteins with antithrombotic and anticoagulant activities 27 and the synthesis of homogeneous polyubiquitin chains by Brik 28 and Liu. 29,30 While the power and utility of chemical protein synthesis is unquestioned, it is often still a tedious process and the construction of most proteins (>100 residues) demands multi-segment ligation strategies that require orthogonal protecting groups, which can complicate the preparation of the constituent peptide segments or interfere with post-ligation processing steps.…”