Sortase A is a transpeptidase that cleaves at a pentapeptide-motif and subsequently transfers the acyl component to a nucleophile containing N-terminal oligoglycines. We investigate the reaction conditions of the sortase-mediated ligation and demonstrate a useful application by the synthesis of a peptide nucleic acid-cell-penetrating peptide chimera, the reaction equilibrium of which can be shifted in favor of the product by dialyzing out the low molecular weight byproduct. The synthesized conjugate exhibits dose-dependent antisense activity.