HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

Abstract

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics

“…This is an order of magnitude faster than previously reported for an equivalent ligation of similarly sized domains 25 . The speed of the reaction is in fact similar to what we observed for short peptides in the presence of MPAA additive and TCEP 31 . The use of HPLC allowed an accurate monitoring of the reaction, including the concentration of the MPAA-activated thioester, which is the most reactive thioester species in the mixture.…”

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP

“…6a). This acid treatment is compatible with most other PTMs besides ubiquitylation such as phosphorylation 44 . Although there has been a focus towards developing what are considered milder deprotection conditions we observed no problems with this acid deprotection and the improvement in installation and ligation of the auxiliary more than compensated for any theoretical disadvantage.…”

“…Peptide synthesis. Peptides were prepared by standard automated solid-phase synthesis using DIC/HOBt activation for Fmoc/t-Bu chemistry (CS Bio 336 automated synthesizer) except for LAPAG-MPAL prepared by HF-free Boc chemistry 44 . Couplings of amino acids were carried out with a five-fold excess of activated amino acid for a minimum of 45 min.…”

Photo‐Triggered Fluorometric Hydrophobic Benzyl Alcohol for Soluble Tag‐Assisted Liquid‐Phase Peptide Synthesis

“…As eparatea pproachi nvolves the use of tertbutoxycarbonyl (Boc)-protected amino acids, but this method requirest he use of "acid-resistant" supports because the Boc group is deprotected using an acid (typically TFA), and ah azardous equipment-demanding acidic deprotection (typically using hydrogen fluoride;H F) is neededt oc leave the peptide from the solid support. [3] The Fmoc solid phase method is therefore mostc ommonly used for the chemical synthesis of peptides. However,b ecause as olid phase methods till has potential limitations, such as an incompatibility with heterogeneous catalysts, al iquid phase methodw ould be better suited for achieving the current goals of peptide synthetic chemistry, namely,t of ine-tune the molecular structure of the designed peptide.…”

“…[6g] Although HF is not required, ah arsh acidic condition is still required and thus milder tag cleavage conditions are preferable. In addition, acidic colorimetric reactions are possible using the HBA tag (1) and the 2,4,5-substitutedH BA tag (3). These tags provide easily distinguishable purple and blue color peptides olutions and peptides pots on thin layer chromatography (TLC) plates, enablingq uantitative assay of the peptide-tag intermediate product.…”