Protein synthesis with the ketoacid-hydroxylamine (KAHA) ligation

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The ability to chemically synthesize biologically active proteins in a controlled fashion is one of the greatest achievements of synthetic chemistry in the last 20 years. As part of these efforts, our group reported the α-ketoacid–hydroxylamine (KAHA) ligation as a chemoselective coupling of large, unprotected peptide segments. The KAHA ligation employs C-terminal peptide α-ketoacids (KAs) and N-terminal peptide hydroxylamines (HAs), which react chemoselectively to form amides or esters. This reaction proceeds in aqueous media without reagents or catalysts.

By developing new building blocks and chemical reactions, we can now easily prepare large peptide segments (40+ residues) bearing the necessary reaction partners – C-terminal peptide α-ketoacids and N-terminal peptide hydroxylamines – by standard Fmoc-SPPS. Along with innovative, orthogonal protecting group strategies, we can easily iterate segment ligations for the synthesis of protein targets up to about 200 residues. Recent examples of proteins prepared with the KAHA ligation include Pup, CspA, UFM1, SUMO2, SUMO3, S100A4, Nitrophorin 4 and betatrophin(lipasin).

Our ability to prepare synthetic proteins with any kind of unnatural amino acids, post-translational modification, fluorescent tag, or synthetic handle forms the basis of our increasing efforts in chemical biology and the synthesis of synthetic proteins of therapeutic interest.

Selected publications:

(6) Bode, J. W.; Fox, R. M.; Baucom, K. D. "Decarboxylative Condensations of alpha-Ketoacids and N-Alkylhydroxylamines: A New Amide Ligation Reaction" Angew. Chem. Int. Ed. 2006, 45, 1248–1252.

(17) Ju, L.; Lippert, A. R.; Bode, J. W. "Stereoretentive Synthesis and Chemoselective Amide-Forming Ligations of C-Terminal Peptide alpha-Ketoacids" J. Am. Chem. Soc. 2008, 130, 4253–4255.

(23) Ju, L.; Bode, J. W. "A general strategy for the preparation of C-terminal peptide a-ketoacids by solid phase peptide synthesis" Org. Biomol. Chem. 2009, 7, 2259–2264.

(49) Wu, J.; J. Ruiz-Rodríguez; Comstock, J. M.; Dong, J. Z.; Bode, J. W. "Synthesis of human GLP-1 (7–36) by chemoselective α-ketoacid–hydroxylamine peptide ligation of unprotected fragments" Chem. Sci. 2011, 2, 1976–1979.

(54) Pattabiraman, V.; Bode, J. W. “Rethinking Amide Bond Synthesis”(Review) Nature 2011, 480, 471–479.

(62) Pattabiraman, V. R.; Ogunkoya, A. O.; Bode, J. W. "Chemical Protein Synthesis by Chemoselective α-Ketoacid–Hydroxylamine (KAHA) Ligations with 5-Oxaproline" Angew. Chem. Int. Ed. 2012, 51, 5114–5118.

(72) Ogunkoya, A. O.; Pattabiraman, V. R.; Bode, J. W. "Sequential α-Ketoacid-Hydroxylamine (KAHA) Ligations: Synthesis of C-Terminal Variants of the Modifier Protein UFM1" Angew. Chem. Int. Ed. 2012, 51, 9693–9697.

(98) Wucherpfennig, T. G.; Rohrbacher, F.; Pattabiraman, V. R.; Bode, J. W. "Formation and Rearrangement of Homoserine Depsipeptides and Depsiproteins in the α-Ketoacid–Hydroxylamine Ligation with 5-Oxaproline" Angew. Chem. Int. Ed. 2014, 53, 12244–12247.

(99) Wucherpfennig, T. G.; Pattabiraman, V. R.; Limberg, F. R. P.; Ruiz-Rodríguez, J.; Bode, J. W. "Traceless Preparation of C-Terminal α-Ketoacids for Chemical Protein Synthesis by α-Ketoacid–Hydroxylamine Ligation: Synthesis of SUMO2/3" Angew. Chem. Int. Ed. 2014, 53, 12248–12252.

(102) Murar, C. E.; Thuaud, F.; Bode, J. W. "KAHA Ligations that form Aspartyl Aldehyde Residues as Synthetic Handles for Protein Modification and Purification" J. Am. Chem. Soc. 2014, 136, 18140–18148.

(105) Harmand, T. J.; Kulkarni, S. S.; Bode, J. W. "Optimized Synthesis of a Cyanosulfurylide Linker for Fmoc-SPPS of C-Terminal Peptide α-Ketoacids" Tetrahedron Lett. 2015, 56, 3477–3480.

(110) Rohrbacher, F.; Deniau, G.; Luther, A.; Bode, J. W. "Spontaneous Head-to-Tail Cyclization of Unprotected Linear Peptides with the KAHA Ligation" Chem. Sci. 2015, 6, 4889–4896.

(115) He, C.; Kulkarni, S. S.; Thuaud, F.; Bode, J. W. "Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation" Angew. Chem. Int. Ed. 201554, 12996–13001.

(121) Harmand, T. J.; Murar, C. E.; Bode, J. W.; "Protein chemical synthesis by α-ketoacid-hydroxylamine ligation" Nature Protocols 201611, 1130-1147

(122) Thuaud, F.; Rohrbacher, F.; Zwicky, A.; Bode, J. W.; "Incorporation of Acid-Labile Masking Groups for the Traceless Synthesis of C-Terminal Peptide α-Ketoacids" Org. Lett. 201618, 3670–3673

(123) Wucherpfennig, T. G.; Müller, S; Wolfrum, C.; Bode, J. W.; "Chemical Synthesis of the 12 kDa Human Myokine Irisin by α-Ketoacid-Hydroxylamine (KAHA) Ligation" Helv. Chim. Acta 2016, Accepted

 
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25.05.2017
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