Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media.

Andreas C Baumruck,Jie Yang,Gerke-Fabian Thomas,Daniel Tietze,Luisa I Beyer,Alesia A Tietze

doi:10.1021/acs.joc.0c02498

Andreas C Baumruck, Jie Yang + Show 4 more

Open Access

https://doi.org/10.1021/acs.joc.0c02498

Copy DOI

Abstract

The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C2mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several “gold-standard” ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0–7.5, resulting in ligation yields of up to 80–95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current “gold-standard” NCL methods.

Highlights

Ionic liquids (ILs) have been shown to be a good alternative to organic solvents for biotransformation reactions.[1−4] Several groups describe the use of ionic liquid (IL) as advantageous solvents for protein refolding because of the ability of ILs with nucleophilic anions to either break or lower the formation of hydrogen bonds in solution, which leads to the suppression of aggregation in proteins.[5−8] The use of 1-methoxyethyl-3methylimidazolium hexafluorophosphate with a water content of 3% was described to be suitable for enzymatic peptide synthesis of tripeptide (ZTyrGlyGlyOEt), resulting in a higher enzyme activity and reaction yield in comparison to conventional systems.[9]
The native chemical ligation (NCL) of membrane-associated peptides by the reaction of a thioester-forming and a cysteine-containing peptide was successfully performed in IL [C2mim][OAc]
We were able to establish an efficient IL-based ligation buffer system which shows equal or even slightly better ligation yields compared to that of standard ligation buffers commonly used in modern peptide chemistry

Summary

Introduction

Ionic liquids (ILs) have been shown to be a good alternative to organic solvents for biotransformation reactions.[1−4] Several groups describe the use of ILs as advantageous solvents for protein refolding because of the ability of ILs with nucleophilic anions to either break or lower the formation of hydrogen bonds in solution, which leads to the suppression of aggregation in proteins.[5−8] The use of 1-methoxyethyl-3methylimidazolium hexafluorophosphate with a water content of 3% was described to be suitable for enzymatic peptide synthesis of tripeptide (ZTyrGlyGlyOEt), resulting in a higher enzyme activity and reaction yield in comparison to conventional systems.[9]. The yield of the ligation product decreased with time due to side reactions, such as oxidative folding or succinimide formation.[16] A somewhat different NCL strategy was reported by Duan et al using hexamethyldisiloxane in [BMIM]PF6 or [BMIM]BF4 as a promoter to ligate a cysteine-free peptide fragment to a thioester peptide.[17] The ligation yields for di-, tri-, and tetrapeptides achieved by this method varied between 60 and 93%. We have found conditions under which the IL can be used as a solvent or as a reactant through the presence of N-heterocyclic carbenes in the neat IL.[18] Based on these recent achievements, we sought to apply IL as reaction media for reactions which are problematic in conventional solvents, that is, conjugation of hydrophobic peptides or orthogonal modifications of peptides. The presented work will focus on the NCL of highly hydrophobic peptides in order to improve the synthetic accessibility to membrane proteins or fragments derived thereof, which in turn would allow for a more detailed structural and functional analysis of, for example, ligand-

Objectives

Results

Conclusion