Improved chemical synthesis of hydrophobic Aβ peptides using addition of C‐terminal lysines later removed by carboxypeptidase B

Abstract

Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid phase synthesis of the Alzheimer’s plaque peptide Aβ40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aβ42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid phase synthesis of Aβ peptides. In our method, Lys residues are linked to the desired peptide’s C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B. With this method we obtained both Aβ42 and Aβ46 of superior quality that, for Aβ42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with carboxypeptidase B removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aβ46, which is extended from the sequence Aβ42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aβ42 or Aβ40. The enhanced amyloidogenicity of Aβ46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aβ amyloid formation.

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

“…Our retrosynthetic strategy involved two peptide segments, with a single ligation junction at position Leu 41 -Ala 42 (Figure 4a, b). Following ligation of the GroES-N and GroES-C peptides, desulfurization (Cys→Ala) at position 42 would generate full-length native GroES.…”

“…We considered introducing an additional ligation site C-terminal to Leu 41 in order to break up this difficult segment. However, the absence of any Cys or Ala residues in this region (residues 43 – 97) complicated this prospect.…”

“…An approach using the acid-labile phenylacetamido (PAM) linker has also been described 40 . Alternatively, linkers containing substrate sequences cleaved by HIV-protease 32 and carboxypeptidase 41 have also been used. Thus far, these base-, acid- and enzyme-sensitive linkers have been limited to peptide termini.…”

C-Terminal Threonine Reduces Aβ43 Amyloidogenicity Compared with Aβ42

“…There have been only limited studies, however, on the solution and assembly properties of longer Aβ species either alone or in mixture with the more common Aβ species. Aβ 46 has been reported to spontaneously aggregate much more rapidly than Aβ 42 and Aβ 40 19 , and mature fibrils of Aβ 46 were reported to seed elongation of Aβ 42 and Aβ 40 20 . Aβ 43 in isolation has been reported to aggregate at similar 21 or faster 22, 23 rates compared with Aβ 42 , and, when present in 10% the molar amount of Aβ 42 , to not alter aggregation kinetics 21 .…”

Medicinal Chemistry Focusing on Aggregation of Amyloid-β

“…12,13) Insufficient peptide coupling-deprotection reactions during solid phase peptide synthesis (SPPS) occurs owing to the aggregation of the on-resin constructing Aβ chain. Upon purification using HPLC after SPPS, the aggregation of Aβ1-42 obstructs the elution of the peptide from the reverse-phase column, resulting in a broad, inseparable peak.…”