Synthesis and Application of Unprotected Cyclic Peptides as Building Blocks for Peptide Dendrimers

Abstract

We describe an efficient regiospecific method for cyclization of unprotected peptide segments based on intramolecular transthioesterification of unprotected cysteinyl peptide thioesters under the control of ring−chain tautomeric equilibrium in aqueous buffered solutions at pH ranging from 5 to 7.5. The initial cyclization to form an intramolecular thioester under the ring−chain tautomeric equilibrium is reversible and could be performed in relatively high concentrations without observable oligomerization. This method overcomes the limitation of conventional cyclization methods that require high dilutions. The reaction becomes irreversible by a subsequent, spontaneous proximity-driven S- to N-acyl transfer to the adjacent N α-amine of cysteine to form an end-to-end cyclic peptide. The cyclization is regioselective. No side reactions were observed with side-chain functionalities such as the N ε-amine of lysine, thiol of internal cysteine, or imidazole of histidine. Since a free thiol group was introduced to the product after cyclization, these cyclic peptides were exploited as building blocks for synthesizing peptides with unusual architectures such as bicyclic peptides containing end-to-end backbones and disulfide bridges as well as cascade branched peptide dendrimers.

Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1

“…The Cterminal segment of Ras, comprising amino acids 118-166, was synthesized on a standard -OCH 2 -Pam resin. The second segment [Ras-(51-117)] was S-Acm-protected at its N-terminal cysteine residue to prevent cyclization (23). The first native chemical ligation of Ras-(51-117) and Ras-(118-166) was carried out at the Lys-117-Cys-118 site in 6 M guanidine hydrochloride buffer at pH 7.0.…”

A biomimetic strategy in the synthesis and fragmentation of cyclic protein

“…was generally similar to that previously described by our laboratory for the thioester ligation of two unprotected peptide segments (Tam et al, 1995;Zhang & Tam, 1997). In the one-pot reaction, the crude peptides cleaved from the resin support by high HF were extracted by 8 M urea solution at pH 7.5 in a highly reductive environment containing a 5-10-fold excess of TCEP (Bums et al, 1991) to prevent disulfide formation.…”

Total synthesis by modern chemical ligation methods and high resolution (1.1 Å) X‐ray structure of ribonuclease A

“…Internal peptides (i.e. 26-39, 40-64, 65-83, 84-94) that contained both an N-terminal Cys and a C-terminal a thioester had their N-terminal cysteine protected as a (4R)-1,3-thiazolidine-4-carboxylic acid (Thz) 32 to prevent undesired side-product formation from cyclization 33 and oligomerization under ligation conditions. To overcome limited solubility, the peptide Thz 65 -83-CO a thioester was synthesized with a hydrophilic tag containing six arginine residues in the thioester leaving group.…”