Discrete Cu(i) complexes for azide-alkyne annulations of small molecules inside mammalian cells

Abstract

The archetype reaction of "click" chemistry, namely, the copper-promoted azide-alkyne cycloaddition (CuAAC), has found an impressive number of applications in biological chemistry. However, methods for promoting intermolecular annulations of exogenous, small azides and alkynes in the complex interior of mammalian cells, are essentially unknown. Herein we demonstrate that isolated, well-defined copper(i)-tris(triazolyl) complexes featuring designed ligands can readily enter mammalian cells and promote intracellular CuAAC annulations of small, freely diffusible molecules. In addition to simplifying protocols and avoiding the addition of "non-innocent" reductants, the use of these premade copper complexes leads to more efficient processes than with the alternative, in situ made copper species prepared from Cu(ii) sources, tris(triazole) ligands and sodium ascorbate. Under the reaction conditions, the well-defined copper complexes exhibit very good cell penetration properties, and do not present significant toxicities.

Fig. 1. (a) Outline of a metal-promoted bimolecular coupling of exogenous molecules in living cells; (b) CuAAC reaction between anthracenyl azide 1 and propargyl alcohol 2.

Chart 1. (a) Structure of water soluble tris(triazolylmethyl)amine ligands; (b) preformed, isolated Cu(i) complexes.

Fig. 2. (a) Yields of the CuAAC with in situ preformed copper complexes. Reaction conditions: CuSO₄ (75 μM) was mixed with 2 equiv. of the ligands L1–L4 in H₂O and the mixture was added to another solution containing anthracenyl azide 1 (100 μM) and propargyl alcohol 2 (200 μM) in either H₂O or PBS. Then, NaAsc was added (2.5 mM) and the reaction was maintained at 25 °C; (b) yields of the CuAAC with the preformed copper complexes C1–C4 (75 μM), using the above concentrations of reactants, with/without sodium ascorbate (2.5 mM), at 37 °C, 20 min; (c) conversion profiles of the CuAAC with different Cu(i) complexes in PBS, in reactions carried out with 25 μM of the copper species. The reaction yields were calculated using a fluorescence calibration curve that was obtained with increasing concentrations of the triazole 3, from 0 to 100 μM.

Fig. 3. Fluorescence micrographies in experiments carried out in HeLa cells using the in situ made copper(i) complexes: 75 μM CuSO₄, 2 equiv. L4, 6 equiv. NaAsc. (A, B) Cells incubated with azide 1 (100 μM) and alkyne 2 (200 μM) for 1 h, followed by double washing with DMEM (2 × 5 min). (C, D) Cells after incubation with the copper containing mixture (30 min), DMEM washings (2 × 5 min), and treatment with 1 (100 μM) and 2 (200 μM) for 1 h, followed by double washing with DMEM (2 × 5 min). (E) Zoom of panel D. Basal levels of fluorescence were normalized by LUT equalization. Scale bar, 12.5 mm. (A and C and brightfield).

Fig. 4. (a) Fluorescence micrographies resulting from the CuAAC reactions with the in situ made Cu(i)/L3 species and with C5, using HeLa cells. (A, D) Cells incubated only with azide 1 (100 μM) and alkyne 2 (200 μM) for 1 h, followed by double washing with DMEM (2 × 5 min). (B, E) Cells after incubation with in situ made copper species with L3, using standard ascorbate reducing conditions (75 μM, 30 min incubation), DMEM washings (2 × 5 min), and treatment with 1 (100 μM) and 2 (200 μM) for 1 h, followed by double washing with DMEM (2 × 5 min). (C, F) Results using complex C5. A 10 mM solution of C5 in DMSO was freshly prepared in an open flask, and used immediately with no further precautions. Cells after incubation with C5 (50 μM, 30 min incubation), DMEM washings (2 × 5 min), and treatment with 1 (100 μM) and 2 (200 μM) for 1 h, followed by double washing with DMEM (2 × 5 min). Basal levels of fluorescence were normalized by LUT equalization. Scale bar, 12.5 mm; (b) flow cytometry analysis for the quantification of fluorescent cells after the reactions promoted by copper complexes. The results with L1–L3 refer to the copper-promoted reactions using these ligands and NaAsc (standard conditions). (A, B and C are brightfield).

Fig. 5. (a) ICP-MS results of the intracellular accumulation of copper after incubation of cells in DMEM with 75 μM of copper complexes (in DMSO) for 2 h, double washing with PBS (2 × 5 min) and digestion with HNO₃. Note that when indicating L2, L3 or L4, the results refer to the copper accumulation using these ligands and NaAsc (standard conditions); (b) viability assays with in situ made Cu(i) complexes with L3, and with preformed C5 for 2 h and 24 h; the amount of viable cells was analysed by MTT assays.