ABSTRACT
Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc-based solid-phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid-phase peptide synthesis, via an NâS acyl shift that is initiated by the presence of a C-terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C-terminal cysteine residue with selenocysteine and initiating thioester formation via an NâSe acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N-terminal cysteine and C-terminal selenocysteine.
Subject(s)
Esters/chemistry , Peptides/chemical synthesis , Selenocysteine/chemistry , Sulfhydryl Compounds/chemistry , Molecular Structure , Nitrogen/chemistry , Peptides/chemistry , Temperature , Time FactorsABSTRACT
Tagging the terminus: NâS acyl transfer in native peptides and proteins can be reliably intercepted with hydrazine. The method allows selective labeling and ligation, without recourse to the use of protein-splicing elements. NCL=native chemical ligation.
Subject(s)
Cysteine/chemistry , Hydrazines/chemistry , Peptides/chemistry , Proteins/chemistry , Amino Acid Sequence , Erythropoietin/chemistry , Glycopeptides/chemistry , Hepcidins/chemistry , Humans , Molecular Sequence Data , Ubiquitin/chemistryABSTRACT
Peptide thioesters are important tools for the total synthesis of proteins using native chemical ligation (NCL). Preparation of glycopeptide thioesters, that enable the assembly of homogeneously glycosylated proteins, is complicated by the perceived fragile nature of the sugar moiety. Herein, we demonstrate the compatibility of thioester formation via NâS acyl transfer with native N-glycopeptides and report observations that will aid in their preparation.