The ‘O-acyl isopeptide method’ for the synthesis of difficult sequence-containing peptides: application to the synthesis of Alzheimer's disease-related amyloid β peptide (Aβ) 1-42

Abstract

An efficient 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides was applied successfully to the synthesis of amyloid beta peptide (Abeta) 1-42 via a water-soluble O-acyl isopeptide of Abeta1-42, i.e. '26-O-acyl isoAbeta1-42' (6). This paper describes the detailed synthesis of Abeta1-42 focusing on the importance of resin selection and the analysis of side reactions in the O-acyl isopeptide method. Protected '26-O-acyl isoAbeta1-42' peptide resin was synthesized using 2-chlorotrityl chloride resin with minimum side reactions in comparison with other resins and deprotected crude 26-O-acyl isoAbeta1-42 was easily purified by HPLC due to its relatively good purity and narrow elution with reasonable water solubility. This suggests that only one insertion of the isopeptide structure into the sequence of the 42-residue peptide can suppress the unfavourable nature of its difficult sequence. The migration of O-acyl isopeptide to intact Abeta1-42 under physiological conditions (pH 7.4) via O--N intramolecular acyl migration reaction was very rapid and no other by-product formation was observed while 6 was stable under storage conditions. These results concluded that our strategy not only overcomes the solubility problem in the synthesis of Abeta1-42 and can provide intact Abeta1-42 efficiently, but is also applicable in the synthesis of large difficult sequence-containing peptides at least up to 50 amino acids. This synthesis method would provide a biological evaluation system in Alzheimer's disease research, in which 26-O-acyl isoAbeta1-42 can be stored in a solubilized form before use and then rapidly produces intact Abeta1-42 in situ during biological experiments.

Specific Recognition of Biologically Active Amyloid-β Oligomers by a New Surface Plasmon Resonance-based Immunoassay and an in Vivo Assay in Caenorhabditis elegans

“…For the present studies we used the "depsi-peptide" technique for A␤ synthesis (9,10), improved in our laboratory (11,12), which consistently produces seed-free starting solutions. This is a prerequisite for any analysis of the properties of highly aggregating peptides (13).…”

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

“…10 More recently, hexapeptides derived from Aβ C-terminal residues 32-37 methylated at multiple positions were soluble and nontoxic and were shown to inhibit the aggregation and toxicity of full-length Aβ40 and Aβ42. 9 N-Methylation of at least two residues of the minimum-length fibril-forming sequence of IAPP, IAPP (22)(23)(24)(25)(26)(27), was sufficient to block β-structure, fibril formation, and peptide toxicity, as demonstrated by Kapurniotu et al 8 The N-methylated amino acids were incorporated in an i, i + 2 arrangement such that lateral β-sheet extension via hydrogen bonding was blocked on one face of the peptide. The same group later used this approach to generate a non-amyloidogenic derivative of full-length IAPP doubly N-methylated at residues G-24 and I-26 with a 100-fold increase in water solubility compared Fig.…”

“…A key advantage of O-acyl or S-acyl isopeptide units over pseudoproline building blocks is that they are stable following standard peptide cleavage methods with TFA and can thus further facilitate purification and handling beyond the synthesis stage. Furthermore, many N(Y) protecting groups have been explored, and a variety of triggering systems such as pH, [22][23][24][25][26] enzymes, 27 reducing agents, 28 and light 29,30 have been developed for restoring the native peptide backbone, some of which are even compatible with in vivo studies (vide infra).…”

“Click Peptide“: pH‐Triggered in Situ Production and Aggregation of Monomer Aβ1–42

“…Namely, modification of the O-acyl-isomerized structure between Gly25-Ser26 of Ab1-42 resulted in an efficient synthesis of 26-O-acyl isoAb1-42 (1; Scheme 1). [21] Due to the ionized N a -amino group of Ser26, 1 was 100-fold more water-soluble than Ab1-42. [21,22] Consequently, 1 was easily purified by HPLC.…”

“…[21] Due to the ionized N a -amino group of Ser26, 1 was 100-fold more water-soluble than Ab1-42. [21,22] Consequently, 1 was easily purified by HPLC. Moreover, Ab1-42 could then be obtained from 1 through a pH-dependent, O-to-N intramolecular acyl migration under neutral conditions (Scheme 1).…”