Polyproline sequences are highly abundant in prokaryotic and eukaryotic proteins, where they serve as key components of secondary structure. To date, construction of the proline-proline motif has not been possible owing to steric congestion at the ligation junction, together with an n -> pi* electronic interaction that reduces the reactivity of acylated proline residues at the C-terminus of peptides. Here, we harness the enhanced reactivity of prolyl selenoesters and a trans-gamma-selenoproline moiety to access the elusive proline-proline junction for the first time through a diselenide-selenoester ligation-deselenization manifold. The efficient nature of this chemistry is highlighted in the high-yielding one-pot assembly of two proline-rich polypeptide targets, submaxillary gland androgen regulated protein 3B and lumbricin-1. This method provides access to the most challenging of previously intractable peptide and protein targets of increasing structural ligation junctions, thus enabling the construction of complexity.