Analogous to self-assembly in natural DNA or proteins, we describe the synthesis of a heterofunctional artificial tripeptide that self-assembles into an antiparallel duplex by coordination of three Cu(II) ions. The tripeptide contains three pendant ligands, pyridine (py), methyl bipyridine (bpy), and terpyridine (tpy), in series on an aminoethylglycine (aeg) backbone. These ligands chelate three Cu(II) ions, forming two [Cu(tpy)(py)](2+) and one [Cu(bpy)(2)](2+) complexes, that cross-link two tripeptide strands to give a trimetallic supramolecular structure. The tripeptide and metal-linked tripeptide duplex are characterized with NMR spectroscopy, mass spectrometry, and analytical high performance liquid chromatography (HPLC). Spectrophotometric titrations are used to quantitatively examine the stoichiometry of binding. Together with electron paramagnetic resonance (EPR) spectroscopy, the identities of the Cu(II) complexes and their environments are examined. The EPR spectrum reveals a significant amount of coupling between metal centers compared to a dimetallic dipeptide analogue. EPR and UV-vis absorbance spectroscopy, together with molecular modeling, provide evidence that the tripeptide acts as a scaffold to hold the metal centers in close proximity.