Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.

Cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-Stokes Raman scattering (CARS) microscopy without the need for labels. Here we review the application of CARS microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. High-resolution CARS microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. The CARS imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules.

HMA2 is a Zn2؉ -ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn 2؉ efflux. Distinct from P 1B -type ATPases, plant Zn 2؉ -ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn 2؉ K1 ⁄ 2 for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn 2؉ with high affinity (K d ‫؍‬ 16 ؎ 3 nM). Circular dichroism spectral analysis indicated the presence of 8% ␣-helix, 45% ␤-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% ␣-helix, 39% ␤-sheet, and 52% random coil , Cd 2ϩ , Pb 2ϩ , Co 2ϩ ) across biological membranes (1-3). These enzymes play critical roles in maintaining heavy metal homeostasis in organisms ranging from bacteria to humans (4 -8). Plant genomes appear to contain multiple (eight or nine) genes encoding P 1B -ATPases with various metal selectivities (Zn 2ϩ -ATPases, Cu ϩ -ATPases, and others with metal dependence is still to be determined) (3,8,9). Distinctly, only two Cu ϩ -ATPase isoforms are found in other eukaryotes (1-3). We recently characterized the functional role of Arabidopsis thaliana HMA2 (10). This Zn 2ϩ -ATPase drives the efflux of metals out of the cell and is activated by Zn 2ϩ and Cd 2ϩ with quite low apparent affinities (0.1-0.2 M). Analysis of A. thaliana hma2 knock-out mutants revealed a significant increase in whole plant Zn 2ϩ and Cd 2ϩ levels (10). This observation along with the plasma membrane localization and strong expression in the plant vasculature suggests that HMA2 is responsible for Zn 2ϩ uploading into the phloem (10, 11). P 1B -type ATPases have 6 -8 transmembrane fragments responsible for metal translocation and a large cytoplasmic loop involved in ATP binding and hydrolysis (1-3). Conserved residues in transmembrane fragments H6, H7, and H8 participate in metal coordination during transport and provide signature sequences that predict the metal selectivity of P 1B -type ATPases (3, 12). Most of these enzymes also have highly conserved N-terminal metal binding domains (N-MBDs) 2 characterized by the CXXC sequences (3, 6, 7, 13). These Cys residues are responsible for metal coordination, and can bind both monovalent and divalent cations (Cu ϩ , Cu 2ϩ , Zn 2ϩ , Cd 2ϩ ) (14 -19). In Cu ϩ -ATPases, N-MBDs receive the metal from specific Cu ϩ -chaperones (20 -26). Removal of the N-MBDs metal binding capability by truncation or mutation leads to reduced enzyme activity with small or no changes in metal affinity (27-33). Lutsenko and co-workers (34) have shown the Cu ϩ -dependent interaction of Wilson's disease protein N-MBDs with the large ATP binding cytoplasmic loop. In our laboratory, we have observed that N-MBDs of Archaeoglobus fulgidus CopA, a Cu ϩ -ATPase, and CopB, a Cu 2ϩ -ATPase with a His-rich N-MBD, control the turnover rate of these enzymes but do not aff...

The addition of Cu(I) to the random-coil peptide, C16C19-GGY, produces a self-organized, metal-bridged 4-helix bundle which displays an intense room-temperature luminescence at 600 nm. Emission, UV, and CD titrations along with X-ray absorption studies indicate that the luminescent cofactor is likely a Cu4S4 cluster in which each Cu atom is bridged by the side chains of two cysteine residues and has terminal N/O ligation.