X‑ray Absorption Spectroscopy Proves the Trigonal-Planar Sulfur-Only Coordination of Copper(I) with High-Affinity Tripodal Pseudopeptides

Abstract

A series of tripodal ligands <b>L</b> derived from nitrilotriacetic acid (NTA) and extended by three converging metal-binding cysteine chains were previously found to bind selectively copper­(I) both in vitro and in vivo. The ligands <b>L</b>^<b>1</b> (ester) and <b>L</b>^<b>2</b> (amide) were demonstrated to form copper­(I) species with very high affinities, close to that reported for the metal-sequestering metallothioneins (MTs; log <i>K</i>^Cu‑MT ≈ 19). Here, an in-depth study by Cu K-edge X-ray absorption spectroscopy (XAS) was performed to completely characterize the copper­(I) coordination sphere in the complexes, previously evidenced by other physicochemical analyses. The X-ray absorption near-edge structure (XANES) spectra shed light on the equilibrium between a mononuclear complex and a cluster for both <b>L</b>^<b>1</b> (ester) and <b>L</b>^<b>2</b> (amide). The exclusive symmetric CuS₃ geometry adopted in the mononuclear complexes (Cu–S ≈ 2.23 Å) was clearly demonstrated by extended X-ray absorption fine structure (EXAFS) analyses. The EXAFS analyses also proved that the clusters are organized on a symmetric CuS₃ core (Cu–S ≈ 2.26 Å) and interact with three nearby copper atoms (Cu---Cu ≈ 2.7 Å), consistent with the Cu₆S₉-type clusters previously characterized by pulsed gradient spin echo NMR spectroscopy. XAS data obtained for other architectures based on the NTA template (<b>L</b>^<b>3</b> acid, <b>L</b>^<b>4</b> without a functionalized carbonyl group, etc.) demonstrated the formation of polymetallic species only, which evidence the necessity of the proximal ester or amide group to stabilize the CuS₃ mononuclear species. Finally, XAS was demonstrated to be a powerful method to quantify the equilibrium between the two copper­(I) environments evidenced with <b>L</b>^<b>1</b> and <b>L</b>^<b>2</b> at different copper concentrations and to determine the equilibrium constants between these two complexes