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