Synthesis and secondary structural studies of penta(acetyl-Hmb)A beta(1-40).
The Fmoc solid phase synthesis of A beta(1-40), a strongly aggregating peptide found in Alzheimer's disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N(alpha)-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using 'semi-on-line' MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native A beta(1-40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of A beta(1-40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)A beta(1-40) was found to adopt both alpha-helix and intermolecular beta-sheet conformations. In 100% TFE a mixed alpha-helix/random coil structure was formed by the protected peptide indicating reduced alpha-helical propensity relative to A beta(1-40). The protected peptide formed beta-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native A beta(1-40), penta(acetyl-Hmb)A beta(1-40) did not form large aggregate species.
School of Chemistry and Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville, Victoria, Australia.
A B
AB
Clippingdale
M
M
Macris
J D
JD
Wade
C J
CJ
Barrow
D000215:Acylation; D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry*; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40)