title: Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation.
abstract: Established methodology for the preparation of peptide thioesters requires the use of t-butoxycarbonyl chemistry owing to the lability of thioester linkers to the nucleophilic reagents used in Fmoc solid phase peptide synthesis. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc-based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non-nucleophilic base, 1,8-diazabicyclo[5.4.0]undec-7-ene, together with 1-hydroxybenzotriazole in dimethylformamide, were used as the N(alpha)-Fmoc deprotection reagent. Native chemical ligation of the resulting thioester product to an N-terminal cysteine-containing peptide was successfully performed in aqueous solution to produce a fragment peptide of human alpha-synuclein. The formation of aspartimide (cyclic imide) in a base-sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, this could be controlled by the judicious protection of sensitive residues using the 2-hydroxy-4-methoxybenzyl group.
authors.affiliation: Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Victoria, Australia.
authors.forename: A B
authors.initials: AB
authors.lastname: Clippingdale
authors.forename: C J
authors.initials: CJ
authors.lastname: Barrow
authors.forename: J D
authors.initials: JD
authors.lastname: Wade
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D004126:Dimethylformamide / Q000737:chemistry; D004952:Esters / Q000138:chemical synthesis*; D005449:Fluorenes / Q000737:chemistry*; D006801:Humans; D008956:Models, Chemical; D009419:Nerve Tissue Proteins / Q000737:chemistry; D010452:Peptide Biosynthesis; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051843:Synucleins; D013997:Time Factors; D014230:Triazoles / Q000494:pharmacology; D051844:alpha-Synuclein
chemical_list: D000596:Amino Acids; D004952:Esters; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009419:Nerve Tissue Proteins; D010455:Peptides; C497604:SNCA protein, human; D051843:Synucleins; D014230:Triazoles; D051844:alpha-Synuclein; D001224:Aspartic Acid; C075871:aspartimide; D004126:Dimethylformamide; C011852:1-hydroxybenzotriazole