Chemical Protein Synthesis with the α-Ketoacid–Hydroxylamine Ligation

Abstract

The coupling of an α-ketoacid and a hydroxylamine (KAHA ligation) affords amide bonds under aqueous, acidic conditions without the need for protecting groups or coupling agents. Translating this finding into a general approach to chemical protein synthesis required the identification of methods to incorporate the key functional groups into unprotected peptide segments-ideally using well-established Fmoc solid-phase peptide synthesis protocols. A decade of effort has now led to robust, convenient methods for preparing peptides bearing free or masked C-terminal α-ketoacids and N-terminal hydroxylamines. The facile synthesis of the segments and the aqueous, acidic conditions of the KAHA ligation make it ideal for the construction of small proteins (up to 200 residues), including SUMO and related modifier proteins, betatrophin and other protein hormones, nitrophorin 4, S100A4, and the cyclic protein AS-48. Key to the successful development of this protein synthesis platform was the identification and gram-scale synthesis of (S)-5-oxaproline. This hydroxylamine monomer is completely stable toward standard methods and practices of solid-phase peptide synthesis while still performing very well in the KAHA ligation. This reaction partner-in contrast to all others examined-affords esters rather than amides as the primary ligation product. The resulting depsipeptides often offer superior solubility and handling and have been key in the chemical synthesis of hydrophobic and ampiphilic proteins. Upon facile O-to-N acyl shift, peptides bearing a noncanonical homoserine residue at the ligation site are formed. With proper choice of the ligation site, the incorporation of this unnatural amino acid does not appear to affect the structure or biological activity of the protein targets. The development of the chemical methods for preparing and masking peptide α-ketoacids and hydroxyalmines, the preparation of several protein targets by convergent ligation strategies, and the synthesis of new hydroxylamine monomers affording either natural or unnatural residues at the ligation site are discussed. By operation under acidic conditions and with a distinct preference for the ligation site, these efforts establish KAHA ligation as a complementary method to the venerable native chemical ligation (NCL) for chemical protein synthesis. This Account documents both the state of the KAHA ligation and the challenges in identifying, inventing, and optimizing new reactions and building blocks needed to interface KAHA ligation with Fmoc solid-phase peptide chemistry. With these challenges largely addressed, peptide segments ready for ligation are formed directly upon resin cleavage, facilitating rapid assembly of four to five segments into proteins. This work sets the stage for applications of the KAHA ligation to chemical biology and protein therapeutics.

Untapped Opportunities of Resin‐to‐Resin Transfer Reactions (RRTR) for the Convergent Assembly of Multivalent Peptide Conjugates

“…The use of precisely addressable transacylation systems, applied in fine‐tuned combinations for the sequential, convergent or iterative assembly of polypeptide molecules, demonstrates the attained level of sophistication. The role of the native(‐type) chemical ligation method(s) confirms the particular desire for the original amide bond, ubiquitous in life and nature, with special focus towards conceptual advances in amidation tactics …”

Chemoselective Derivatization of Folded Synthetic Insulin Variants with Potassium Acyltrifluoroborates (KATs)

“…We initiated the synthesis of the ornithine hydroxylamine building block (4) with the reduction of the commercially available 2-nitroacetophenone (Scheme 2,b). The resulting racemic alcohol (S1) was isolated on a gram scale and activated with N,N'-disuccinimidyl carbonate followed the procedure of Thuaud et al [19] Hydroxylamine hydrochloride was N-acylated utilizing the activated carbonate (1), prepared in the previous step, and O-acylated using N-diethylcarbamoyl chloride to give hydroxylamine 2 with the appropriate functionalities. Fmoc-Glu(OH)O t Bu was transformed to the corresponding side-chain alcohol (3) in excellent yield.…”

“…In contrast, the photocaged variant proved to be completely stable to all standard manipulations for resin cleavage, peptide purification, assembly with KAHA ligation, Acm deprotection with silver, and protein folding. This finding allowed facile construction of synthetic folded Glargine M2 insulin variant by KAHA ligation [19] and its conjugation to a variety of KATs suitable for protein labeling, PEGylation, lipidation, and dimerization.…”

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

“…To expand the scope of NCL, a series of advanced versions of NCL have been developed for ligation at non‐Cys residues, such as Ala, Lys, Val, Ser, and Thr . Meanwhile, some non‐NCL methods have also been developed, such as serine/threonine ligation (STL) and α‐keto acid–hydroxylamine (KAHA) ligation, to enable peptide ligation at non‐Cys sites. These non‐NCL methods have been used to synthesize a number of proteins (e.g.…”