Chemical synthesis of a homoserine-mutant of the antibacterial, head-to-tail cyclized protein AS-48 by α-ketoacid–hydroxylamine (KAHA) ligation

Rohrbacher, Florian; Zwicky, André; Bode, Jeffrey W.

doi:10.1039/c7sc00789b

Cited by 39 publications

(27 citation statements)

References 42 publications

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“…Many bioactive cyclic peptides have been synthesized by this strategy, such as antibiotic gramicidin S (Figure A), tyrocidine A (Figure B) and antineoplastic stylostatin A (Figure C) . In 2017, a head‐to‐tail cyclic antibacterial protein, named AS‐48, was also successfully prepared by two KAHA ligations of three peptide segments …”

Section: Strategies To Develop Cyclic Peptides Into Therapeutic Agentsmentioning

confidence: 99%

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

Jing

Jin

2019

Medicinal Research Reviews

122

130

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As a versatile therapeutic modality, peptides attract much attention because of their great binding affinity, low toxicity, and the capability of targeting traditionally “undruggable” protein surfaces. However, the deficiency of cell permeability and metabolic stability always limits the success of in vitro bioactive peptides as drug candidates. Peptide macrocyclization is one of the most established strategies to overcome these limitations. Over the past decades, more than 40 cyclic peptide drugs have been clinically approved, the vast majority of which are derived from natural products. The de novo discovered cyclic peptides on the basis of rational design and in vitro evolution, have also enabled the binding with targets for which nature provides no solutions. The current review summarizes different classes of cyclic peptides with diverse biological activities, and presents an overview of various approaches to develop cyclic peptide‐based drug candidates, drawing upon series of examples to illustrate each strategy.

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Section: Strategies To Develop Cyclic Peptides Into Therapeutic Agentsmentioning

confidence: 99%

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

Jing

Jin

2019

Medicinal Research Reviews

122

130

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“…This is demonstrated by synthesis of the poorly soluble therapeutic lipopeptide tesamorelin and variants of the transmembrane lipoprotein phospholemman FXYD1 using this method and nanomolar concentrations circumventing the integration of solubilizing units (Chisholm et al, 2019). Broadening the ligation toolkit, the α-ketoacidhydroxylamine (KAHA) ligation route is also employable to poorly soluble and highly hydrophobic proteins such as IFITM3 (see Table 1) or the antibacterial cyclic AS-48 protein (Rohrbacher et al, 2017) making use of 5-oxaproline within acidic conditions. The KAHA ligation is applicable to synthesized fragments from Fmoc-synthesis, integrated solubilizing tags and based on ligation conditions in organic solvents and thus presents an alternative to fragments facing solubility problems.…”

Section: Hot Topics and Outlookmentioning

confidence: 99%

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

Mueller

Baumruck

Zhdanova

et al. 2020

Front. Bioeng. Biotechnol.

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Solid phase peptide synthesis (SPPS) provides the possibility to chemically synthesize peptides and proteins. Applying the method on hydrophilic structures is usually without major drawbacks but faces extreme complications when it comes to "difficult sequences." These includes the vitally important, ubiquitously present and structurally demanding membrane proteins and their functional parts, such as ion channels, G-protein receptors, and other pore-forming structures. Standard synthetic and ligation protocols are not enough for a successful synthesis of these challenging sequences. In this review we highlight, summarize and evaluate the possibilities for synthetic production of "difficult sequences" by SPPS, native chemical ligation (NCL) and follow-up protocols.

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“…Photolabile protecting groups were installed onto both segments, which were joined by KAHA ligation in AcOH/HFIP -a modification necessary to solubilize the difficult segments and promote their union -to give the ester product. 22 Photochemical removal of the protecting groups followed KAHA cyclization site KAHA ligation site by cyclization of the unfolded protein gave the bis-depsipeptide in decent yield. Upon exposure to folding conditions, this peptide underwent two acyl shifts -one fast, one slow -to give the folded, cyclic peptide with a 11-minute shift on the HPLC!…”

Section: Synthesis Of the Cyclic Protein As-48mentioning

confidence: 99%

Chemical Protein Synthesis with the α-Ketoacid–Hydroxylamine Ligation

2017

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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.

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Chemical synthesis of a homoserine-mutant of the antibacterial, head-to-tail cyclized protein AS-48 by α-ketoacid–hydroxylamine (KAHA) ligation

Abstract: The development of ligation conditions suitable for highly hydrophobic peptide segments allowed the first chemical synthesis of the head-to-tail cyclized protein AS-48.

Cited by 39 publications

References 42 publications

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

Chemical Protein Synthesis with the α-Ketoacid–Hydroxylamine Ligation

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