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Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation
Author(s) -
Clippingdale Andrew B.,
Barrow Colin J.,
Wade John D.
Publication year - 2000
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/(sici)1099-1387(200005)6:5<225::aid-psc244>3.0.co;2-t
Subject(s) - thioester , native chemical ligation , chemistry , peptide , peptide synthesis , chemical ligation , nucleophile , combinatorial chemistry , protecting group , reagent , solid phase synthesis , cysteine , chemical synthesis , imide , organic chemistry , stereochemistry , biochemistry , in vitro , alkyl , enzyme , catalysis
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 α ‐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 α‐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. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.