Using hydroxymethylphenoxy derivates with the SPOT technology to generate peptides with authentic C-termini

The SPOT technology can fulfill most requirements for highly parallel, multiple peptide synthesis of soluble peptides within the upper microgram range. Here, we report on an improved method using hydroxymethylphenoxyacetic acid (HMPA) for 19 amino acids and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) for proline as acidic labile linkers in SPOT synthesis. Using this approach we could reduce side-chain reactions normally occurring during conventional alkaline peptide cleavage from cellulose membranes. All synthesis steps were adapted to fully-automated SPOT synthesis and therefore represent a time- and cost-saving procedure. Furthermore, the improved cleavage and washing steps resulted in peptides with authentic C-termini in a purity range of 60–95%. Our improved method is ideal for synthesizing many thousand different peptides subsequently used directly for different biological assays requiring authentic C-termini, such as CD8 T-cell epitope screening, vaccine immunization, or tumor imaging.