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.