title: Automated synthesis of a C-terminal photoprobe using combined Fmoc and t-Boc synthesis strategies on a single automated peptide synthesizer.
abstract: Peptide photoprobes can be utilized to identify peptide binding sites of various proteins. The photoprobe p-benzoylbenzoic acid (BBA) possesses several unique chemical characteristics which make it ideal for the construction of a peptide photoprobe: It can be directly coupled to the peptide during synthesis, it is stable to HF treatment, it is inert until photolysis, and the photo-activated probe is non-specific in its binding to proteins. The construction of an N-terminal p-benzoylbenzoyl-peptide photoprobe utilizing an automated peptide synthesizer is straight-forward because p-benzoylbenzoic acid can be incorporated during routine peptide synthesis as if it were the N-terminal amino acid. In contrast, the construction of a C-terminal photoprobe peptide is more complex and requires the presence of a C-terminal lysine to incorporate the photoprobe into the peptide. We have devised a suitable pathway to attach BBA to the C-terminal of a synthetic peptide in an automated fashion. The method described in this report utilizes Fmoc chemistry for the automated loading of Boc-Lysine-epsilon-Fmoc to hydroxymethyl resin. After loading, the epsilon-amine of the resin-bound lysine is deprotected with piperidine and the addition of BBA to the epsilon-amine then occurs via a peptide bond. The peptide synthesizer is then converted over to t-Boc chemistry, and the remainder of the synthesis is carried out using standard protocols.
authors.affiliation: HHMI Core Protein/Peptide Facility Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: J
authors.initials: J
authors.lastname: Gorka
authors.forename: D W
authors.initials: DW
authors.lastname: McCourt
authors.forename: B D
authors.initials: BD
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002621:Chemistry / Q000379:methods; D007202:Indicators and Reagents; D008239:Lysine; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis*; D010782:Photolysis; D012116:Resins, Plant; D012710:Serum Albumin, Bovine
chemical_list: D007202:Indicators and Reagents; D010455:Peptides; D012116:Resins, Plant; D012710:Serum Albumin, Bovine; D008239:Lysine