Hydrazide-Mediated Solubilizing Strategy for Poorly Soluble Peptides Using a Dialkoxybenzaldehyde Linker

• Published in: Chemical & pharmaceutical bulletin Vol. 70; no. 10; pp. 707 - 715
• Main Authors: Tanaka, Shoko, Narumi, Tetsuo, Mase, Nobuyuki, Sato, Kohei
• Format: Journal Article
• Language: English
• Published: TOKYO The Pharmaceutical Society of Japan 01.10.2022; Pharmaceutical Soc Japan; Japan Science and Technology Agency
• Subjects: Alkylation; Chemical compounds; chemical protein synthesis; Chemical synthesis; Chemistry; Chemistry, Medicinal; Chemistry, Multidisciplinary; Fluorophores; Hydrazides; Hydrophilicity; Intermediates; Life Sciences & Biomedicine; peptide hydrazide; Peptides; Pharmacology & Pharmacy; Physical Sciences; poorly soluble peptide; Protecting groups; Protein biosynthesis; Protein synthesis; Proteins; Science & Technology; Solubility; Solubilization; solubilizing tag; Structural analysis; Structure-function relationships; Trifluoroacetic acid; Ubiquitin; TOTAL CHEMICAL-SYNTHESIS; poorly soluble peptide; LIGATION; peptide hydrazide; TAG; solubilizing tag; MEMBRANE-PROTEINS; chemical protein synthesis; ubiquitin; GENERAL-METHOD
• ISSN: 0009-2363; 1347-5223
• DOI: 10.1248/cpb.c22-00501

Proteins modified in a controlled manner with artificial moieties such as fluorophores or affinity tags have been shown to be a powerful tool for functional or structural analysis of proteins. A reliable way to prepare proteins with a well-defined modification is protein synthesis. Although many successful syntheses have been reported, the poor aqueous solubility of synthetic intermediates causes difficulty in the chemical synthesis of proteins. Here we describe a solubilizing strategy for poorly soluble peptides which uses chemoselective incorporation of a hydrophilic tag onto a hydrazide in a peptide. We found that a hydrophilic tag possessing a dialkoxybenzaldehyde moiety can react with peptide hydrazides through reductive N-alkylation. No protecting groups are required for this reaction, and peptides modified in this way show enhanced solubility and consequently good peak separation during HPLC purification. The tag can be removed subsequently by treatment with trifluoroacetic acid to generate a free hydrazide, which can be converted in a one-pot reaction to a thioester for further modification. This method was validated by synthesis of a Lys63-linked ubiquitin dimer derivative. This late-stage solubilization can be applied in principal to any peptide and opens the possibility of the synthesis of proteins that have previously been considered inaccessible due to their poor solubility.