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

Cu(i) complexes do achieve azide–alkyne annulations of freely difusible small molecules inside mammalian cells.


Introduction
Organometallic catalysis has changed the eld of organic synthesis in the last half century, and has found important applications in other areas such as materials, energy or environmental sciences. In spite of such wide impact, the use of transition metal catalysis in biological contexts remains underdeveloped, probably due to the general belief that metalpromoted reactions are incompatible with the air atmospheres and aqueous environments of biological habitats, and that the metal complexes can be highly cytotoxic. 1 Only recently, a few examples demonstrating the viability of achieving transition metal promoted transformations in biological contexts, 2 and even in intracellular environments, 3 have been disclosed. Most of these reports deal with palladium or ruthenium-catalyzed uncaging of designed substrates equipped with inactivating handles. 4 More challenging intracellular metalpromoted coupling reactions involving two different abiotic precursors are much scarcer. This is not surprising, as these reactions require the cell entrance and "meeting" of up to three different partners, namely the metal complex and two exogenous reactants (Fig. 1a). Thus, while several groups have demonstrated the viability of achieving Suzuki or Sonogashira couplings on appropriately modied proteins in E. coli, 5 the only two examples described so far in mammalian cells involve the use of palladium nanoparticles, and xed cells. Curiously, the well-known copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction, 7 has been very scarcely explored inside the complex environment of living mammalian cells. Thus, while early work on the use of this reaction for biological purposes was restricted to bacteria, 8 most of the other "in vivo" applications have been limited to the modication of cell surface labelled glycans. 9 Probably, the established notion that copper is highly cytotoxic, and the requirement of excess of several additives, including "non-innocent" ascorbate, have precluded further research to implement the reaction in the challenging and crowded atmosphere of mammalian cells. 9d,10 In recent years, several groups have developed water soluble ligands for Cu(I) that accelerate the reaction and also act as sacricial scavengers/reductants of the reactive oxygen species (ROS) generated by copper, decreasing its cellular toxicity. 11 The accelerating effect can be further improved if the copper chelated ligand is covalently linked to the azide, a tactic that has even been used for the intracellular labelling of proteins. However, with this strategy, the copper complex is likely sequestered by the products, owing to the presence of the triazole and the copper chelating moiety. 12 Water soluble copper ligands linked to a cell penetrating peptide have been recently used to promote click reactions inside cells, but with low efficiency, and only for alkyne-modied proteins. 10b If the modication of intracellular proteins is a highly relevant, and far from trivial, goal, achieving intracellular copper promoted reactions between two "freely diffusing small molecules" is even more challenging. Acceding to this type of reactivity can open new, exciting opportunities for biological or metabolic intervention, and for a metal-dependent generation of active drugs or optical signals. To the best of our knowledge, the only example of such type of intracellular CuAAC reaction relies on the use of cross-linked copper containing polymers termed metalorganic nanoparticles (MONPs), and requires high concentration of sodium ascorbate. 13 Another class of copper nanostructures that can also promote the reaction in water has been recently reported, however their activity is conned to the extracellular milieu. 14 Herein we report the rst examples of an intracellular CuAAC transformation involving two exogenous, freely spreading substrates (small molecule azide and alkyne), promoted by discrete Cu(I) complexes (Fig. 1b). We also present data on the compared reactivity, redox stability, cell uptake and toxicity of in situ made copper species versus Cu(I) predened complexes. These studies allow for the discovery of an independently isolated, well-dened Cu(I) complex equipped with the BTTE ligand (3-4-{{bis{[1-(1,1-dimethylethyl)-1H-1,2,3-triazol-4-yl] methyl}amino}methyl}-1H-1,2,3-triazole-1-ethanol, L3), which performs much better than the in situ mixture obtained from the ligand, a Cu(II) source and sodium ascorbate.

Results and discussion
Our work was conceived on the hypothesis that designed, well-dened Cu(I) complexes might cross cell membranes and keep their oxidation +1 state under the reductive atmosphere of the cell. Thus we proposed to study the intracellular reactivity of tris-triazolyl-Cu(I) complexes generated in situ by reduction of Cu(II) precursors with ascorbate, as well as of isolated, well-dened Cu(I) complexes (Chart 1). 15 As substrates we chose uorogenic azides that undergo an increase in uorescent emission upon annulation with the corresponding alkynes. 16 The most habitual azide substrate for these purposes is 3-azido-7-hydroxycoumarin, however, in our hands, preliminary control tests with HeLa cells indicated that this azide presents a substantial background signal. We therefore moved to the 9-(azidomethyl)anthracene (1, Fig. 1b) which is almost non-uorescent, but undergoes a ca. 150-fold increase in uorescence upon its annulation with alkynes ( Fig. S2 †). This increment can be explained in terms of suppression of the internal PET (photoinduced electron transfer) quenching on moving from the azide to the triazole structure. 16,17 As tris(triazolylmethyl)amine ligands we selected BTTAA (3-4-{{bis{[1-(1,1-dimethylethyl)-1H-1,2,3-triazol-4-yl]methyl}amino}-methyl}-1H-1,2,3-triazole-acetic acid, L1), which has been shown to be rather effective in CuAAC in aqueous media, and even in E. coli. 9c We also prepared the analogues BBTE (L2) and BTTE (L3), which feature a hydroxyl group susceptible of conjugation to different units. Indeed, we synthesized the derivative L4 which contains a triphenylphosphonium moiety designed to favor cellular internalizations and, eventually, mitochondrial localizations (Chart 1a). As predened Cu(I) catalysts, we initially aimed to explore several previously characterized species such as pyrazolyl, NHC (N-heterocyclic carbene) phosphite or phosphinite copper complexes (C1-C4, Chart 1b). It is surprising that the catalytic activity of this type of well-dened Cu(I) complexes had never been explored in biorelevant settings.
Before moving to cellular environments, we investigated the performance of the above complexes in aqueous media. With ligands L1-L4, the catalytic reactions were carried out using 75 mol% of copper, by mixing CuSO 4 with 2 equiv. of the ligand in water (with 2% DMSO) at room temperature for 10 min, and adding the solution to either water or PBS (phosphate buffered solution) mixtures of anthracenyl azide 1 (100 mM) and propargyl alcohol 2 (200 mM), followed by sodium ascorbate (NaAsc, over 30 equiv.).
For comparison purposes, we analyzed the conversion aer 10 and 20 min, by using calibration curves (see Fig. 2 and Section S4 in the ESI †).
In the absence of ligands, i.e., when only CuSO 4 and sodium ascorbate are employed, the reaction proceeds with poor yields (<10% even aer 24 h, Fig. 2a, dark blue bars). However, with ligand L3 the product was obtained in 32% yield in water and 22% in PBS, aer 10 min, while L2 was less effective. Notably, using the phosphonium containing ligand L4 we observed 50% of the triazole aer 10 min, in both water and PBS (phosphatebuffered saline, Fig. 2a, purple bars), and a very good 70% yield aer 20 min. As expected, if we skip the pre-treatment of the Cu(II) complexes with sodium ascorbate, there is no reaction. UV-Vis and 1 H-NMR analysis conrmed that mixing CuSO 4 , the ligand and sodium ascorbate generates a tris(triazole) Cu(I) species ( Fig. S9 and S10 †).
The performance of the predened, isolated Cu(I) complexes C1-C4 (Chart 1b) was also assessed in the absence or presence of ascorbate, at 37 C (20 min, Fig. 2b). The carbene complex C3 is almost inactive, and the phosphite and phosphinite complexes C2 and C4 also led to very poor conversions (less than 2% of the product). With the complex [Cu(NCMe)(Tpa*)] [PF 6 ] (C1) 15a,b the reaction was slightly more efficient (13% in water and 5% in PBS). Importantly, addition of sodium ascorbate allowed much better conversions, specially, with C2 and C3. These results suggest that under the reaction conditions (open air ask), the Cu(I) species are readily oxidized, something that was further conrmed by EPR. Therefore, while C1 and C2 are stable in solid state, in DMSO they are very rapidly oxidized under air to give paramagnetic Cu(II) species (Fig. S11 and S12 †).
Overall, the best conversions were achieved with the in situ made copper complexes in presence of ligand L4. Indeed, using this ligand it was possible to obtain the product in a satisfactory 46% yield, aer 20 min, using just 25 mM of the copper source (Fig. 2c). 18 With the above information in hand, we moved to living mammalian cells using two different cell lines: HeLa and A549 (living human cervical cancer cells and adenocarcinomic human alveolar basal epithelial cells, respectively). In the experiments with sodium ascorbate, the copper containing mixture added to the cells was prepared by mixing CuSO 4 and the ligand (L) in a 1 : 2 ratio in water for 1 h, followed by treatment with an aqueous solution of sodium ascorbate (6 equiv.) for 30 min. 19 With the dened, discrete Cu(I) species C1-C4, cells were directly incubated with a freshly made DMSO solution of the complexes. The experiments were carried out by mixing cultured cells with the copper solutions (75 mM for in situ made complexes and 50 mM for discrete Cu(I) species) for 30 min in fresh DMEM (Dulbecco's modied Eagle's medium), followed by two washing steps with DMEM prior to the addition of the reactants. The resulting cells were incubated with the azide 1 (100 mM) and the alkyne 2 (200 mM) in fresh DMEM for 60 min and washed twice with DMEM, before observation under the uorescence microscope. It is important to note that we do not use cell xation techniques, which allows for the preservation of the native living environment, and avoids artefacts or over-interpretations.
In the experiments with in situ made copper species, in absence of ligands or with L2, we did not detect any intracellular uorescence, while with L1 and L3 the uorescent intensity was weak (Fig. S18 †). However, we were glad to observe that when using L4 as ligand, there was a clear blue intracellular uorescence across the cytoplasm and in vesicles, with the cells showing an unaltered morphology (Fig. 3,

panel C, D and E).
Control experiments in absence of the copper species (Fig. 3, panel A and B), using the same threshold observation parameters, conrm that the signal must necessarily come from the expected reaction. 20,21 Remarkably, despite their low in vitro activity, the predened Cu(I) complex C1, the phosphite complex C2 and the phosphinite complex C4 were able to raise some intracellular uorescence in experiments carried out in the absence of ascorbate, while C3 failed to elicit any uorescence (Fig. S19 †).
Using MTT cytotoxicity assays we observed that more than 90% of the cells survived aer 2 h of treatment with the standard Cu(II)/L4/ascorbate mixture, using 75 mM of the copper Fig. 2 (a) Yields of the CuAAC with in situ preformed copper complexes. Reaction conditions: CuSO 4 (75 mM) was mixed with 2 equiv. of the ligands L1-L4 in H 2 O and the mixture was added to another solution containing anthracenyl azide 1 (100 mM) and propargyl alcohol 2 (200 mM) in either H 2 O 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 mM), 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 mM 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 mM. source (80% survival aer 12 h). With C1 the cell survival was slightly lower, reaching values of approx. 80% aer 2 h (Fig. S22 †).
The reactivity observed with the tris(pyrazolyl) copper species C1, prompted us to pursue the specic preparation of a well-dened Cu(I) complex equipped with a tris(triazole) ligand. Thus, we focused on the isolation of a complex similar to C1 but containing the ligand L3 or L4. While with L4 we have not yet been successful, we could isolate a Cu(I) complex (C5) by mixing [Cu(NCMe) 4 ][PF 6 ] with equimolar amounts of L3 in methanol, and subsequent precipitation (Section S2 †).
EPR monitoring of fresh DMSO solutions of this complex (C5) demonstrated a higher redox stability than C1. Therefore, while in the case of C1, 80% of Cu(I) is oxidized to Cu(II) aer 20 min, under the same conditions, less than 30% of C5 was oxidized ( Fig. S12 and S13 †). The in vitro performance of complex C5 was quite similar to that of tris(pyrazolylmethane)containing complex C1, however we were pleased to observe that this complex presents an excellent performance in native cellular settings, in the absence of sodium ascorbate (Fig. 4a, panel C and F); much better than that observed when the cells are incubated with the standard pre-made mixture containing Cu(II)/L3/ascorbate (Fig. 4a, panel B and E).
To better appreciate the differences in efficiency, we have established a protocol to calculate reaction yields of the intracellular transformations, based on uorescence measurements using a microplate reader (see Section S13 in the ESI †). The data were normalized with respect to the amount of anthracenyl azide (1, limiting reactant) uptaken by cells. Gratifyingly, when complex C5 was used, the product was obtained in approx. 18% yield, which is over 7 times greater than that obtained using the in situ prepared complex with ligand L3.
The intracellular reactivity was also analyzed by ow cytometry, which conrmed that cells treated with C5 presented higher levels of uorescence when compared with that resulting from the in situ made L3/copper complex. Indeed, C5 performed the best among all the copper species so far studied (Fig. 4b). The use of an extensive washing protocol to remove extracellular copper should assure that the reactions are taking place inside the cells. However, we further conrmed this by observing a total lack of reactivity in control experiments using extracellular media. Furthermore, we also observed that adding copper chelators like EDTA to the extracellular solution, in experiments carried out with living cells, has no effect on the results (see Fig. S20 †).
Interestingly, there is a clear correlation between the copper uptake and the observed activity. Therefore, the phosphonium containing ligand L4 promoted a relatively high intracellular accumulation of copper. The ICP-MS analysis also indicates that the well-dened Cu(I) complexes C1 and C2 are very well internalized, which explains why we do observe some intracellular reactivity despite their poor in vitro activity. More important, the copper complex C5 is also very well internalized, leading to almost three times more internal copper than that from the corresponding in situ made copper complex with the ligand L3.  Therefore, the better intracellular performance of C5 versus the in situ made mixture of CuSO 4 /L3/NaAsc appears to be, at least in part, associated to an improved internalization.
Cell toxicity studies using different concentrations of C5, indicated over 70% viability aer 2 h, which is raised to 82% using 25 instead of 50 mM ( Fig. 5b and S22 †). If we normalize these values with respect to the amount of copper internalized by cells (ICP data) we can conclude that the toxicity of complex with L3, and complex C5 is similar. An additional control experiment indicated that the intracellular reaction is also feasible with 25 mM of C5, albeit the efficiency is slightly lower (over 9-10% yield, page S31).
The above information conrms that the effectivity of an intracellular CuACC with C5 is associated to a good balance between redox stability and catalytic reactivity, and to its improved cell uptake properties, and furthermore conrms the viability of obtaining efficient, well-dened Cu(I) catalysts to be used in complex intracellular environments.

Conclusions
We have demonstrated that water soluble copper(I) complexes featuring designed ligands can readily enter mammalian cells and promote intracellular CuAAC annulations of small, abiotic and freely diffusible molecules.
Our results indicate that using appropriate ligands, it is possible to tune the cell uptake and reactivity of Cu(I) complexes, and importantly, conrm the viability of using discrete copper species to promote efficient CuAAC annulations in the challenging interior of mammalian cells. Indeed, an independently isolated Cu(I)-tris(triazolylmethyl)amine complex, C5, that can be stored without degradation when kept under nitrogen, is capable of promoting the intracellular transformation even in the absence of ascorbate. This new complex displays better cellular uptake and better intracellular reactivity than that observed for the in situ made Cu(I)/L3 complex.
This complex is therefore working as an "off-the-shelve" catalyst to promote challenging intermolecular annulations inside mammalian cells. The copper complex C5 circumvents some of the actual limitations of the "in vivo" CuAAC chemistry, since it avoids the use of excess of ligands or the use of reductants such as ascorbate.
Current studies are focused on further improving the ligands to obtain even more effective catalysts that demonstrate negligible toxicity, on the development of copper complexes that can target different cellular organelles and on the use of the complexes for achieving designed biological alterations.

Conflicts of interest
There are no conicts to declare.  ) for 2 h, double washing with PBS (2 Â 5 min) and digestion with HNO 3 . 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.