Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation

Abstract

Established methodology for the preparation of peptide thioesters requires the use of t-butoxycarbonyl chemistry owing to the lability of thioester linkers to the nucleophilic reagents used in Fmoc solid phase peptide synthesis. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc-based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non-nucleophilic base, 1,8-diazabicyclo[5.4.0]undec-7-ene, together with 1-hydroxybenzotriazole in dimethylformamide, were used as the N(alpha)-Fmoc deprotection reagent. Native chemical ligation of the resulting thioester product to an N-terminal cysteine-containing peptide was successfully performed in aqueous solution to produce a fragment peptide of human alpha-synuclein. The formation of aspartimide (cyclic imide) in a base-sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, this could be controlled by the judicious protection of sensitive residues using the 2-hydroxy-4-methoxybenzyl group.

Advances in Fmoc solid‐phase peptide synthesis

“…In the Boc method, peptide thioesters can be prepared directly [241,242], whereas for the Fmoc method, the use of piperidine at each cycle is not compatible with a thioester at the C-terminus [243,244]. Several indirect methods have therefore been proposed.…”

Tidbits for the synthesis of bis(2‐sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters

“…The use of Fmoc-SPPS and thioester linkers for peptide thioester synthesis is faced with the instability of the thioester group in the presence of the base used for Fmoc removal. One solution to this problem is to decrease the basicity of the mixture used for Fmoc removal with acidic additives such as HOBt [12,13]. Another approach is based on the stabilization of the thioester linker using steric effects [14].…”

Fragment Condensation of C‐Terminal Pseudoproline Peptides without Racemization on the Solid Phase

“…When either DBU or 1-methylpyrrolidine [13] were used, mainly diketopiperazine formation was slowed and the best results were obtained with DBU/HOBt. [14] Fragment B was resynthesized on 2-Cl-Trt resin 4 (Scheme 3). After deprotection of 4 a with DBU/piperidine/ DMF (2:2:96) the third amino acid was coupled.…”