Chemical Protein Synthesis with the KAHA Ligation

Abstract

Since the first report of the chemoselective amide bond forming reaction between α-ketoacids and hydroxylamines in 2006, the KAHA (α-ketoacid-hydroxylamine) ligation has advanced to a useful tool for the routine synthesis of small to medium sized proteins and cyclic peptides. In this chapter we introduce the concept of KAHA ligation starting with the synthesis and properties of hydroxylamines and α-ketoacids, methods for their incorporation into peptides, and give an insight into the mechanism of the KAHA ligation. We cover important improvements including sequential ligations with 5-oxaproline, traceless synthesis of peptide α-ketoacids and show their application in chemical protein synthesis and cyclic peptide synthesis. Recent developments of the KAT (potassium acyl trifluoroborate) ligation and its application as fast and chemoselective bioconjugation method are described and an outlook on ongoing work and possible future developments is given at the end of the chapter.

Chemical Synthesis of the 12 kDa Human Myokine Irisin by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation

“…Our strategy for the synthesis of irisin is based on the assembly of the full‐length protein from smaller, synthetically accessible peptides using chemoselective KAHA ligations. The central motif of the KAHA ligation is the amide bond‐forming reaction between two fully unprotected protein segments bearing a C‐terminal α ‐ketoacid and an N‐terminal hydroxylamine, respectively …”

“…The central motif of the KAHA ligation is the amide bond-forming reaction between two fully unprotected protein segments bearing a C-terminal a-ketoacid and an N-terminal hydroxylamine, respectively. [16] [17] The best implementation of this ligation utilizes a cyclic hydroxylamine, 5-oxaproline (Opr), [18] which has two unusual features: i) The primary ligation product is the ester-containing depsipeptide rather than the expected amide. [19] These depsipeptides, which help improve solubility and handling of the unfolded synthetic proteins, are readily converted to the natural amides using mild basic buffers.…”

KAHA Ligation at Serine

“…Compared with type I, type II KAHA ligation is much more practical for chemical protein synthesis. A number of small‐ to medium‐sized proteins have been synthesized by 5‐Opr‐assisted KAHA ligation …”

Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation

“…[1] Until the advent and adoption of native chemical ligation (NCL), first utilized for protein synthesis by Kent and co-workers in 1994, [2] chemical protein synthesis was restricted to only ahandful of examples requiring Herculean efforts to synthesize longer segments and couple them together. [7] Nevertheless,t he search for thiol-independent chemical reactions suitable for protein synthesis has been very attractive.S everal methods,i ncluding the Staudinger ligation, [8] the a-ketoacid-hydroxylamine(KAHA) ligation, [9] and the serine/threonine ligation of peptide salicylate esters, [10] offer promising advances,b ut none other than NCL has yet [5] Significant progress has been made to address some of the key issues in using NCL, including selective desulfurization reactions [6] and the development of new linkers to prepare peptide thioesters using Fmoc-based solid-phase peptide synthesis (SPPS;F moc = 9-fluorenylmethoxycarbonyl).…”

“…[3] NCL has enabled the preparation of dozens of proteins, [1,4] including examples over 300 residues in length. [7] Nevertheless,t he search for thiol-independent chemical reactions suitable for protein synthesis has been very attractive.S everal methods,i ncluding the Staudinger ligation, [8] the a-ketoacid-hydroxylamine(KAHA) ligation, [9] and the serine/threonine ligation of peptide salicylate esters, [10] offer promising advances,b ut none other than NCL has yet [7] Nevertheless,t he search for thiol-independent chemical reactions suitable for protein synthesis has been very attractive.S everal methods,i ncluding the Staudinger ligation, [8] the a-ketoacid-hydroxylamine(KAHA) ligation, [9] and the serine/threonine ligation of peptide salicylate esters, [10] offer promising advances,b ut none other than NCL has yet…”