Concurrent and orthogonal gold(I) and ruthenium(II) catalysis inside living cells

The viability of building artificial metabolic pathways within a cell will depend on our ability to design biocompatible and orthogonal catalysts capable of achieving non-natural transformations. In this context, transition metal complexes offer unique possibilities to develop catalytic reactions that do not occur in nature. However, translating the potential of metal catalysts to living cells poses numerous challenges associated to their biocompatibility, and their stability and reactivity in crowded aqueous environments. Here we report a gold-mediated C–C bond formation that occurs in complex aqueous habitats, and demonstrate that the reaction can be translated to living mammalian cells. Key to the success of the process is the use of designed, water-activatable gold chloride complexes. Moreover, we demonstrate the viability of achieving the gold-promoted process in parallel with a ruthenium-mediated reaction, inside living cells, and in a bioorthogonal and mutually orthogonal manner.

1 H NMR (300 MHz) spectra were recorded at room temperature on a Varian Mercury 300 MHz spectrometer and 1 H NMR (500 MHz) spectra were recorded at room temperature on a Bruker DRX-500 spectrometer. 13 C NMR (126 MHz) were recorded on a Bruker DRX-500 spectrometer and 13 C NMR (75 MHz) Varian Mercury 300 MHz. 31 P NMR (202 MHz) spectra were recorded on a Bruker DRX-500 spectrometer. 19 F NMR (282 MHz) spectra was recorded on a Varian Mercury 300 MHz spectrometer. The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentuplet, h = septuplet, m = multiplet, br = broad signal, bs = broad singlet. NMR spectra were analyzed using MestreNova© NMR data processing software (www.mestrelab.com). The chemical shifts (δ) are given in ppm and the coupling constant (J) in Hz.
High resolution mass spectra (HRMS) were acquired using electrospray (ESI) and were recorded at the CACTUS facility of the University of Santiago de Compostela.
Measurements of fluorescence were performed using a Varian Cary Eclipse fluorometer. The measurements were made with the following settings: increment 1.0 nm, averaging time 0.1 s, excitation slit width 5.0 nm, emission slit width 5.0 nm, PMT voltage 620 V.
Measurements of UV were performed using a Jasco V-670 spectrometer.

Electrospray Ionization Mass Spectrometry (ESI/MS) was performed with a Bruker Amazon IT/MS using direct injection of a solution of the compound into the MS.
Analytical HPLC was performed, except noted otherwise, on an Agilent 1260 Infinity II coupled to an Agilent Technologies 6120 Quadrupole LC-MS using a flow rate of 0.35 mL/min at room temperature. For the solvent system was used as initial conditions H2O/MeCN (50:50) followed by a gradual change over 35 min to H2O/MeCN (0:100). The chromatogram was recorded via UV absorption at λ = 254 nm.

[AuCl(PPh2Ph(p-CO2H)] (Au3)
Procedure adapted from Monkowius et al. 3 [AuCl(SMe2)] (0.339 mmol, 100.0 mg, 1.0 eq.) and CH2Cl2 (5.0 mL) were successively added to a heat gun dried Schlenk equipped with a stir bar. After the complex was dissolved, 4-(diphenylphosphino)benzoic acid was added (0.339 mmol, 100.0 mg, 1.0 eq.) followed by CH2Cl2 (5.0 mL) and the mixture was stirred at room temperature for 1 h to yield a colorless solution. The solution was then concentrated (to ca. 3.0 mL) in vacuum, and the addition of hexane (ca. 15.0 mL) precipitated a white solid, which was washed with hexane (3 x 15.0 mL) and diethyl ether (3 x 10.0 mL) and dried in vacuum. The gold complex (Au3) was isolated as a white solid and stored under nitrogen.

[AuCl(PPh2Ph(m-SO3Na)] (Au4)
Procedure adapted from Laguna et al. 4 [AuCl(SMe2)] (0.270 mmol, 80.0 mg, 1.0 eq.) and CH2Cl2 (5.0 mL) were successively added to a heat gun dried Schlenk equipped with a stir bar. After the complex was dissolved, 3-(diphenylphosphino) benzenesulfonic acid sodium salt was added (0.270 mmol, 98.0 mg, 1.0 eq.) followed by CH2Cl2 (5.0 mL) and the mixture was stirred at room temperature for 1 h to yield a colorless solution. The solution was then concentrated (to ca. 3.0 mL) in vacuum, and the addition of hexane (ca. 15.0 mL) precipitated a white solid, which was washed with hexane (3 x 15.0 mL) and diethyl ether (3 x 10.0 mL) and dried in vacuum. The gold complex (Au4) was isolated as a white solid and stored under nitrogen. NaAuCl4 (0.10 mmol, 40.0 mg, 1.0 eq.), KPF6 (0.30 mmol, 56.0 mg, 3.0 eq.) and 10.0 mL of H2O were successively added to a round bottom flask equipped with a stir bar. After the solution was complete homogeneous, a solution of phenantroline (0.10 mmol, 18.0 mg, 1.0 eq.) in MeCN (1.0 mL) was added. The resulting solution was refluxed for 15 h and the colorless hot reaction mixture filtered. The filtrate was washed with H2O (5 x 5.0 mL) to ensure the complete removal of any unreacted NaAuCl4 and dried in vacuum. The solid was dissolved in acetone (15.0 mL) and heated at 50 °C. This hot solution was then filtered and concentrated (ca. 3.0 mL). Diethyl ether (15.0 mL) was added and a yellow solid precipitated. After removal of the solvent by decantation, the solid was washed with diethyl ether (3 x 10.0 mL) and dried under vacuum. The gold complex (Au5) was isolated as an orange solid and stored under nitrogen. Yield = 86% (0.086 mmol, 50.0 mg). [AuCl(SMe2)] (0.170 mmol, 50.0 mg) and CH2Cl2 (5.0 mL) were successively added to a heat gun dried Schlenk equipped with a stir bar. After the complex was dissolved, AgNTf2 was added (0.170 mmol, 65.7 mg, 1.0 eq.). The reaction mixture was stirred at room temperature for 2 h in the absence of light. After that, the solution was filtered over Kieselguhr. To the resulting solution, PTA (0.170 mmol, 26.70 mg, 1.0 eq.) was added and the reaction was stirred at room temperature for 2 h. The solution was then concentrated (to ca. 3.0 mL) in vacuum, and the addition of hexane (ca. 15.0 mL) precipitated a white solid, which was washed with hexane (3 x 15.0 mL) and diethyl ether (3 x 10.0 mL) and dried in vacuum. The gold complex (Au1´) was isolated as a white solid and stored under nitrogen.

[Au(NTf2)(PPh2Ph(m-SO3Na)] (Au4´)
Procedure adapted from Gagosz et al. 6 [AuCl(PPh2Ph(m-SO3Na))] (0.050 mmol, 30.0 mg) and CH2Cl2 (15.0 mL) were successively added to a heat gun dried Schlenk equipped with a stir bar. After the complex was dissolved, AgNTf2 was added (0.050 mmol, 19.4 mg, 1.0 eq.) and the mixture was stirred at room temperature in the absence of light for 2 h. After that, the solution was filtered over Kieselguhr to remove AgCl. The solution was then concentrated (to ca. 3.0 mL) in vacuum, and the addition of hexane (ca. 15.0 mL) precipitated a white solid, which was washed with hexane (3 x 15.0 mL) and dried in vacuum. The gold complex (Au4´) was isolated as a white solid and stored under nitrogen.

SUPPLEMENTARY NOTE 4. Details for the gold-catalyzed hydroarylation in water using procoumarin 1.
Substrate 1 (0.050 mmol, 14.69 mg) was added to a Schlenk tube containing a stir bar followed by the addition of the corresponding gold complex (0.0025 mmol, 5 mol%). H2O (1.0 mL) was added and the Thermowatch-controlled heating block was fixed at 37 °C and the reaction was stirred for 24 h. The reaction mixture was extracted with CH2Cl2 (3 x 10.0 mL) and the combined organic fractions were dried, concentrated and the crude was analyzed by 1 H-NMR using CH2Br2 as internal standard.  In order to know if the additives that do not affect the reaction were consumed, the crude reaction of the catalytic hydroarylation in the presence of tyrosine was analyzed. After an extraction with CH2Cl2 (3 x 10.0 mL) and H2O (10 mL), both phases were analyzed: the organic layer concentrates were analyzed by 1 H-NMR spectroscopy, which confirmed the formation of the expected product 4. The residue of the aqueous phase was injected (10 µL These results confirmed that the additives were not consumed or transformed in other products during the catalytic reaction.

Inhibition by adenine
On the other hand, the observed inhibition by adenine was also investigated by ESI-MS. Thus, in a 1.5 mL HPLC vial the gold complex Au4 (0.0008 mmol, 0.50 mg) was dissolved in water (1.0 mL) and then adenine (0.008 mmol, 1.1 mg, 10.0 eq.) was added. After 15 min, the mixture was injected (5 µL) in Bruker Amazon IT/MS. Figure 3. ESI-MS spectra of the reaction between the complex Au4 and adenine.

Supplementary
As can be seen in Supplementary Figure 3, the ESI-MS data confirm that adenine coordinates the gold(I) cation which results from the ionization of Au4 in water (L-Au + species). Thus, two different gold(I)-adenine complexes, depending on whether the sulfinic sodium salt has been protonated (as sulfinic acid) or not, are observed [m/z = 674 and 696].

Representative general procedure for the catalytic hydroarylation using different biological media (exemplified for the use of PBS)
Substrate 4 (0.050 mmol, 15.90 mg) was added to a Schlenk tube containing a stir bar, followed by the addition of [AuCl(PTA)] (Au1, 0.005 mmol, 1.90 mg, 10.0 mol%). MeCN (200 µL) was added and the reaction mixture was stirred at 300 rpm until a homogenous solution was formed (10 s). PBS (800 µL) was added (final volume 1.0 mL, [Substrate] = 50 mM), the Thermowatchcontrolled heating block was fixed at 37 °C and the reaction was stirred for 24 h. After this time, the reaction mixture was extracted with CH2Cl2 (3 x 10.0 mL) and the combined organic fractions were dried to afford the corresponding product 4.

SUPPLEMENTARY NOTE 7. NMR and MS studies on the chloride dissociation in water.
Representative procedure for the 31 P-NMR experiments: All the experiments were performed with the complex Au4 which was added to an NMR tube and dissolved in the corresponding solvent (MeCN or H2O). Then, the capillary tube with the alendronic acid dissolved in D2O (used as reference) was added (see Supplemenry Figure 11). Figure 11. Representative procedure for the NMR experiments.

Monitorization of the chloride dissociation
The complex Au4 was dissolved in 300 µL of H2O and the 31 P-NMR spectra was recorded, observing a signal at 32.96 ppm (Supplementary Figure 12a).
In parallel, we analyzed the samples by ESI-MS: in a 1.5 mL eppendorf the gold complex Au4 was dissolved in H2O and the sample (50 µL) was injected in Bruker Amazon IT/MS. As it can be observed in Supplementary Figure 12b, the sample in pure water shows four different peaks which correspond to aquo species (m/z = 557.0 and 579.0) and chloride complexes (m/z = 596.97 and 618.95). Moreover, a sample in pure MeCN was also injected, and only the latter chloride complex was detected (Supplementary Figure 12c).

Addition of NaCl to the gold complex in water
Increasing amounts of a solution of NaCl (1 M) were added to the NMR tube that contained the solution of Au4 in water, and the 31 P-NMR spectra was recorded. As it is displayed in Supplementary Figure 13a, after sequential addition of 0.5 and 70 equiv. of NaCl, the phosphorus signal shifted from 32.96 ppm to 33.22 and 33.72 ppm, respectively. Finally, when a saturated solution of NaCl was employed as solvent, the signal appeared at 34.06 ppm (all these experiments were performed using alendronic acid as internal standard).
To evaluate whether the change in ionic strength of the solvent (from pure water to saturated NaCl) could be behind of such important shift in the 31 P-NMR (from 32.96 to 33.72 ppm), we measured the 31 P-NMR spectra of the corresponding phosphine ligand [3-(diphenylphosphino) benzenesulfonic acid sodium salt] in water and in a saturated solution of NaCl. As it is displayed in Supplementary Figure 14, no changes in the chemical shift were observed, suggesting that the shift is not due to changes in the ionic strength of the media, but instead must arise from the presence of the above mentioned equilibrium of gold-aquo and gold-chloride species.
To further prove this, the addition of NaCl (aq) to Au4 was also followed by ESI-MS: NaCl was added to a solution of Au4 in H2O to reach a final concentration of NaCl of 5 M. After 5 min, the mixture was injected in the Bruker Amazon IT/MS and we observed that the signal of the gold(I)aquo species (m/z = 557.0 and 579.0) had essentially disappeared, whereas the peak of the goldchloride complex (m/z = 619.0) substantially increased (Supplementary Figure 13b). Interpretation of results from Figure 13 and 14: From these results, we deduce that in a saturated solution of chloride anions the proposed equilibrium between the gold(I)-aquo and gold-chloride species of Au4 is shifted to the latter. Importantly, this hypothesis is consistent with the fact that complex Au4 is not catalytically active in a 6 M solution of NaCl (see main manuscript), whereas is fully active in water (see Supplementary  3 into 4). In other words, in a saturated solution of NaCl, neutral gold(I) chlorides are predominant.

Monitorization of the hydrolysis of Au4
We have also studied by 31 P-NMR and ESI-MS the ionization of Au4 by adding increasing amounts of water to an acetonitrile solution of Au4.
In particular, complex Au4 was dissolved in MeCN (300 µL) and its 31 P-NMR spectra was recorded, observing the expected signal (singlet) at 35.35 ppm (Supplementary Figure 15).
In parallel, we analyzed the samples by ESI-MS: In a 1.5 mL eppendorf the gold complex Au4 was dissolved in MeCN and a sample (50 µL) was injected in Bruker Amazon IT/MS. As it can be observed in Supplementary Figure.  In conclusion, 31 P-NMR and ESI-MS data fully confirm the presence of aquo, acetonitrile and chloride complexes, in different proportions. Moreover, we observe that the presence of water is necessary for the dissociation of the chloride: when Au4 is dissolved in pure MeCN, the ESI-MS only shows the gold chloride complex (m/z = 618.96). Only when water is present, we detect the acetonitrile complex. We propose that water is required for the dissociation of chloride and for triggering the reactivity. We cannot discard associative mechanisms in presence of the alkyne, that further facilitate the dissociation of the chloride in water.

Coordination of piperidine
Similarly to the studies with acetonitrile, we observed that the gold(I) cationic species generated in situ in presence of water could be trapped by nucleophiles such as piperidine. Control experiments confirmed that these species are not formed using MeCN as solvent.
In two separate 1.5 mL HPLC vials, the gold complex Au4 (0.0008 mmol, 0.50 mg) was dissolved in dry MeCN (1.0 mL) or water (1.0 mL) and then piperidine (0.017 mmol, 2 µL, 20 eq.) was added to each vial. After 15 min, the mixture (5 µL) was injected in the Bruker Amazon IT/MS. As can be seen in Supplementary Figure 18, when water is used as solvent three different cationic goldpiperidine complexes are observed, while the chloride-gold complex remains exclusive in MeCN. This confirms the role of water for the ionization of the Au-Cl bond, which must occur prior to the coordination of piperidine. Curiously, the 31 P-NMR spectra of a sample of this water solution displays a single peak, a singlet at 32.96 ppm, suggesting that there is a rapid equilibrium between these gold species. 46 Not surprisingly, when an excess of NaCl is added to the water solution of Au4, the resulting ESI-MS and NMR analysis suggest that the equilibrium is shifted to the chloride complex ( Supplementary   Figure 12a). Furthermore, adding increasing amounts of water to an acetonitrile solution of Au4 leads to the progressive formation of gold(I)-aquo and gold(I)-acetonitrile species (see Supplementary Figure 16 and 17). Also enlightening, ESI-MS analysis of a mixture obtained after addition of 20 equiv. of dry piperidine to an acetonitrile solution of Au4 reveals no reaction after 15 min (only Au4 detected, see Supplementary Figure 18). However, when this experiment is repeated in water, the disappearance of the gold(I)-chloride complex Au4 and the formation of the corresponding gold(I)-piperidine derivative is observed, a result that is consistent with a water promoted dissociation of the chloride atom, and coordination of piperidine to gold (see Supplementary Figure 18).

All together, these data confirm that water promotes the ionization of the Au-Cl bond and
thus drives the complexation of the reactants to the gold(I) complex, which eventually allows to initiate the catalysis.

SUPPLEMENTARY NOTE 9. NMR studies on the solubility of Au6 complex.
An equimolar mixture of Au1 and Au6 was introduced in an NMR tube and DMSO-d6 was used as solvent. 31 P-NMR spectrum shows an approximately 1:1 mixture of both complexes after integration of the signals (Supplementary Figure 20a). In MeCN-d3, the proportion decreases to 1:2 Au1:Au6 (Supplementary Figure 20b). In contrast, in the deuterated mixture of solvents employed for the catalytic reaction, we could only detect the signal of Au1 (Supplementary Figure 20c). This result reflects the insolubility of Au6 in the reaction media. These results confirmed that the lack of reactivity of Au6 in water, and in MeCN/water mixtures (see Supplementary Tables 1 and 2) is due to its lack of solubility, which hinders its waterpromoted activation. In fact, in acetonitrile, where Au6 and the precursor 3 are fully soluble, Au6 does not catalyzed the conversion of 3 into 4, unless a chloride scavenger (such as AgSbF6) is used to generate such ionic gold(I) species (e.g. L-Au + SbF6 -).

SUPPLEMENTARY NOTE 10. General information for the biological experiments.
General executions and substances: All steps were performed on a sterile clean bench Tesltar AV-100 at room temperature. Solutions stored in a fridge were warmed beforehand in a water bath (37 °C). Unless otherwise specified, all incubations were performed in DMEM.
Fluorescence microscopy: All images were obtained with an Andor Zyla mounted on a Nikon TiE. Images were further processed with Image J or NIS software (Nikon).

SUPPLEMENTARY NOTE 11. Viability assays.
The toxicity of the gold complexes was tested by using of the propidium iodide and MTT assays in HeLa cell line.
Propidium iodide assay: 10 15000 cells per well were seeded in 96 well plates one day before treatment. Cells were washed once with KRH buffer. To each well, 100 µL of propidium iodide solution in Krebs-Ringer-Hepes buffer (KRH, 50 µM) were then added. After 20 min of incubation at 37 °C, initial fluorescence from each well was measured in a microtiter plate reading spectrophotometer (Tecan Infinite 200 PRO, λexc = 560 nm). Then, different concentrations of the gold complexes were added. Subsequently, fluorescence was measured every 30 min. Between measurements, microtiter plate was incubated at 37 °C. At the end of the experiment, 100 µL of digitonin in KHR (1 mM) was added to each well to permeabilize all cells and label all nuclei with propidium iodide. Fluorescence was measured again to obtain a value corresponding to 100% cell death.

SUPPLEMENTARY NOTE 12. Experiments in living cells.
HeLa cells growing on glass coverslips were incubated with either catalyst Au1-Au8 (75 µM) for 30 min. Cells were then washed twice with DMEM and incubated with substrate 3 (100 µM) for 6 h. Prior to observation by fluorescence microscopy, the samples were washed twice with fresh DMEM. The coverslips were observed in vivo in a fluorescence microscope equipped with adequate filters. Digital pictures of the different samples were taken under identical conditions of gain and exposure. The calculation was performed on a dual-color images from fluorescent microscopy experiments. These coefficients were calculated with the public domain tool JACoP implemented in the program ImageJ. Mander´s overlap coefficient (MOC) is based on the Pearson´s correlation coefficient but it doesn´t take into account the average intensity values in its mathematical expression. 12 As a result, this parameter is almost independent of signal proportionality and is instead only sensitive to co-ocurrence. MOC varies from 0 to 1, the former corresponding to non-overlapping images and the latter reflecting 100% colocalization between both images. Since MOC is very sensitive to noise, a threshold to the estimated value of background, equalized for every image, was used as zero.

SUPPLEMENTARY NOTE 13. Control reaction between Au1 and substrate 5.
In a Schlenk tube, 100 µL of a solution of substrate 5 (10 mM in MeCN) was added to a 1 mL of H2O : MeCN (8:2) mixture (final concentration: 1 mM). Then, 100 µL of a solution of complex Au1 (10 mM in H2O : MeCN 8:2) was added, the Thermowatch-controlled heating block was fixed at 37 °C and the reaction stirred. The status of the reaction was checked immediately and after 15 h. 100 µL were taken from the solution, diluted with 300 µL of MeCN and filtered through a HPLC-filter in an HPLC vial, and the residue injected in the HPLC Bruker Amazon IT/MS using a flow rate of 0.35 mL/min at room temperature using the gradient H2O/MeCN (95:5) to H2O/MeCN (5:95) over 14 min. The results confirmed that the Au1 cannot promote the deallylation reaction of the substrate 5.

SUPPLEMENTARY NOTE 14. ICP analysis
For the ICP measurements, a total of 3 x 10 6 HeLa cells growing in 6 well plates were treated with 75 µM of the different gold complexes in DMEM for 1 h. Prior to digestion, the samples were washed with fresh DMEM and then twice with PBS. The obtained fractions were digested in duplicate in HNO3/H2O2 by microwave heating and analyzed by ICP-MS.

SUPPLEMENTARY NOTE 15. Flow cytometry studies.
After the incubation time, cells were washed twice with PBS, harvested with trypsin/EDTA for 15 min and resuspended in 2% FBS in PBS buffer with 5 mM EDTA. The fluorescence results of the intracellular reactions were analyzed by flow cytometry. As observed in Figure 4, quantification by flow cytometry showed fluorescence corresponding to the product using the Green-B emission filter (512/18 nm).

SUPPLEMENTARY NOTE 16. Estimation on the turnover number in cells.
CAUTION: These "quantitative" results should be considered just indicative, and not be overinterpreted. Working with millions of living cells that do not always have the same confluence or shape, and using experimental protocols that include washing, extraction or cell counting steps, etc. can introduce significant errors.
In addition, the amount of gold considered is that resulting from ICP-MS measurements, which not necessarily correlates with the real amount of active complex inside cells; and we do not know the amount of substrate that is uptaken by the cells.
General procedure: The hydroarylation reaction of substrate 3 was quantified by fluorescence measurements using a Varian Cary Eclipse fluorometer.
The experiments were performed in plates of 100 mm as follows: 100000 cells per well were seeded in 100 mm plates two days before treatment with the gold(I) complex and the substrate 3. For each measurement, four plates were used.
Cells growing on plate of 100 mm were incubated with Au1 (15 µL, 50 µM) in 3 mL of DMEM for 30 min. Cells were then washed twice with DMEM and incubated with substrate 3 (30 µL, 100 µM) for 6 h. Prior to extraction, the samples were washed with 2 mL of DMEM followed by two washes with PBS (2 mL). Then 1 mL of MeOH in water was added. After 5 min and pipetting up, this solution was transferred to a 1.5 mL eppendorf. Finally, we obtained 5 mL of extracts from the four plates we used.
To normalize the results, the number of cells was measured after the experiments. After the incubation time, cells were washed twice with 2 mL of PBS, harvested with 1 mL of trypsin/EDTA for 5 min at 37 °C and resuspended in 2 mL of DMEM buffer. An aliquot of 50 µL was transferred to a 1.5 mL Eppendorf and diluted with 450 µL of PBS. Then the number of cells was measured with ScepterTM 2.0 Handheld Automated Cell Counter.