Development of a Linker with an Enhanced Stability for the Preparation of Peptide Thioesters and Its Application to the Synthesis of a Stable-Isotope-Labelled HU-Type DNA-Binding Protein

Abstract

An S-alkyl-thioester-moiety-containing linker with an enhanced stability on a resin has been developed. A linker containing S-alkyl thioester with a spacer group, –CO–SC(CH3)2CH2CO–Nle–, was stable during the peptide chain elongation cycle. This thioester was stable under HF treatment conditions, and was rapidly activated by silver ions in the presence of N-hydroxysuccinimde (HONSu) to form a peptide bond. Using this linker, peptide segments covering the HU-type DNA-binding protein of Bacillus stearothermophilus (HBs), which was site-specifically labelled with 2H, 13C, and 15N, were prepared. Using these peptide segments, multi-labelled HBs was synthesized. Distinct signals of 2H, 13C, and 15N in HBs were detected by NMR spectrometry.

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

“…The two segments Ras-(1-50) and Ras-(51-117) were synthesized on a resin designed to generate peptide ␣-thioesters after HF cleavage (16). The Cterminal segment of Ras, comprising amino acids 118-166, was synthesized on a standard -OCH 2 -Pam resin.…”

“…RBD was synthesized from two unprotected peptide segments, RBD-(51-95)␣COSR ϩ RBD-(Cys-96-131), and Ras was synthesized from three segments, Ras-(1-50)␣COSR, Ras-[Cys (Acm) 51-117]␣COSR, and Ras-(Cys-118 -166). Both Nterminal peptide segments and the middle segment Ras-(51-117) were synthesized on a resin that generates a C-terminal thioester after HF cleavage (16). The thiol group of the Nterminal cysteine residue of Ras-(51-117) was protected by the acetamidomethyl (Acm) group, which can be removed by treatment with 1.5 eq of Hg(OAc) 2 per thiol group in a buffer containing 2 M urea at pH 3.5-4 (17).…”

Design, total chemical synthesis, and binding properties of a [Leu-91- N 1 -methyl-7-azaTrp]Ras-binding domain of c-Raf-1

“…In this case, we used the in situ neutralization͞2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate activation protocols for Boc chemistry (28). The N-terminal segments, comprising amino acids 50-95, were synthesized on a resin designed to generate a thioester at the C terminus after HF cleavage (29). The C-terminal fragments, comprising amino acids 96-140 (including the His tag) were synthesized on a standard -OCH 2 -phenylacetamidomethyl resin (33).…”

“…Solid-phase peptide synthesis (27) was performed manually or on a custom-modified 430A peptide synthesizer from Applied Biosystems, using in situ neutralization͞2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate activation protocols for stepwise Boc chemistry chain elongation (28). The N-terminal peptide fragments were synthesized on a thioester-generating resin (29). The coupling of Boc-protected N 1 -methyl-7-azatryptophan was achieved by activation using the 1-hydroxy-7-azabenzotriazole͞diisopropylcarbodiimide method.…”

Synthesis of Native Proteins by Chemical Ligation

“…Despite successful syntheses of the HIV-1 protease (22) and of a limited number of other proteins (30)(31)(32)(33)(34)(35), at the start of the decade of the 1990s total chemical synthesis, by the standard methods of peptide chemistry of even a small protein molecule remained a daunting task, often requiring large teams and taking years to complete, with no guarantee of success. The routine, reproducible preparation of synthetic polypeptides of defined chemical structure was limited to products of ∼50 amino acid residues (19; Figure 2).…”