Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

Full elucidation of the functions and homeostatic pathways of biological copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as Cu . Buffered at attomolar concentrations, intracellular Cu is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, we reveal a chelator design strategy in which phosphine sulfides aid in Cu coordination while simultaneously stabilizing aliphatic phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissociation constant and 10 -fold selectivity for Cu over Zn , Fe , and Mn . As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring Cu in a broad range of biological systems.

Fluorescence probes represent an attractive solution for the detection of the biologically important Cu(I) cation; however, achieving a bright, high-contrast response has been a challenging goal. Concluding from previous studies on pyrazoline-based fluorescent Cu(I) probes, the maximum attainable fluorescence contrast and quantum yield were limited due to several non-radiative deactivation mechanisms, including ternary complex formation, excited state protonation, and colloidal aggregation in aqueous solution. Through knowledge-driven optimization of the ligand and fluorophore architectures, we overcame these limitations in the design of CTAP-3, a Cu(I)-selective fluorescent probe offering a 180-fold fluorescence enhancement, 41% quantum yield, and a limit of detection in the sub-part-per-trillion concentration range. In contrast to lipophilic Cu(I)-probes, CTAP-3 does not aggregate and interacts only weakly with lipid bilayers, thus maintaining a high contrast ratio even in the presence of liposomes.