A Highly Potent Antibacterial Agent Targeting Methicillin-Resistant

Article pubs.acs.org/Organometallics

A Highly Potent Antibacterial Agent Targeting Methicillin-Resistant Staphylococcus aureus Based on Cobalt Bis(1,2-Dicarbollide) Alkoxy Derivative Youkun Zheng,† Weiwei Liu,† Yun Chen,† Hui Jiang,† Hong Yan,‡ Irina Kosenko,§ Lubov Chekulaeva,§ Igor Sivaev,§ Vladimir Bregadze,§ and Xuemei Wang*,† †

State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China ‡ State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China § A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov Str. 28, Moscow 119991, Russia S Supporting Information *

ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is a notorious superbug that is potentially lifethreatening. Among conventional antibiotics, vancomycin is a “gold standard” agent used to treat serious MRSA infections. Such therapy, however, is often ineffective because of the emergence of less-susceptible strains. Therefore, the exploration of new antimicrobial agents, especially nonantibiotic drugs, to cope with the growing threat of MRSA has become an urgent necessity. Herein, we have investigated the possibility to develop a metallacarborane antimicrobial agent, cobalt bis(1,2-dicarbollide) alkoxy derivative (K121), and we have evaluated the relevant anti-MRSA behaviors. We demonstrated that K121 has a dose-dependent anti-MRSA activity with a low minimal inhibitory concentration of 8 μg/mL and a high selectivity over mammalian cells. In particular, a high bacteria-killing efficiency was observed with eradication of all MRSA cells within 30 min. In addition, K121 showed a high inhibition effect on the formation of bacterial biofilm. More importantly, unlike vancomycin, a repeated use of K121 would not induce drug resistance even after 20 passages of MRSA. The mechanistic study showed that K121 kills MRSA by inducing an increase in the reactive oxygen species (ROS) production and consequentially inducing irreversible damage to the cell wall/ membrane, which ultimately leads to the death of MRSA. Our results suggested that K121 may be used as a promising nonantibiotic therapeutic agent against MRSA infections in future clinical practices.

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medicinal chemistry.6−8 To address bacterial drug resistance, the polyhedral pharmacophores-based antimicrobial agents have been widely studied recently. For example, our previous study discovered that ferrocene-carborane derivative could prevent bacterial biofilm formation and damage the bacterial cell wall, with a minimum inhibitory concentrations (MICs) of 36 μg/mL for both multidrug resistance pathogens, S. aureus and Pseudomonas aeruginosa.9 Besides, the antibiotic-resistance reversal by multidrug resistant clinical pathogens has been achieved by using ruthenium(II)-arene complex, a polyhedral carborane.10 It has also been demonstrated that organometallics could efficiently inhibit the activity of β-lactamase and maintain the capability of β-lactam antibiotics to lyse MRSA cells.11 Overall, polyhedral carboranes provide excellent reservoirs for bacterial infection treatments with many advantages owing to their unique properties.12

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA), first discovered in 1961, is an important pathogen that may cause severe infections.1 It represents the capital letter “S” in the most notorious pathogens, ESKAPE, which poses a serious threat to public health especially prevalent in hospitals, and hypervirulent MRSA strains have recently spread throughout the community.2−4 Vancomycin was the first-line drug and “gold standard” agent to treat serious MRSA infections.5 Unfortunately, such therapy has become less effective due to the emergence of lesssusceptible strains. Although several new antibiotic drugs, such as linezolid, daptomycin, and ceftaroline, are also used in the treatment of various MRSA infections, drug resistance and safety remain public health concerns.1 Therefore, the exploration of new antimicrobial agents especially nonantibiotic drugs against MRSA infections is urgently needed. Polyhedral carboranes and especially their metallocomplexes (i.e., metallacarboranes) are a promising class of pharmacophores that have attracted more and more attention in © 2017 American Chemical Society

Received: June 8, 2017 Published: September 1, 2017 3484

DOI: 10.1021/acs.organomet.7b00426 Organometallics 2017, 36, 3484−3490

Article

Organometallics Scheme 1. Synthesis Procedure of K121

Figure 1. Concentration-dependent anti-MRSA activity of K121. (a) Photographs of plates and (b) corresponding statistics of MRSA colonies after treatment with K121 at 0, 10, or 100 μg/mL. (c) Survival rates of MRSA after treatment with K121 at different concentrations. (d) Killing kinetics of K121 at 2× MIC on MRSA.

HIV protease inhibition,24−27 and antibacterial activity,28,29 and were thus considered as a class of drug candidates. In this contribution, we have investigated the antimicrobial activity of a typical bifunctional cobalt bis(1,2-dicarbollide) alkoxy derivative30 (designated as K121, see Scheme 1) as a promising nonantibiotic therapeutic agent against MRSA infections. We demonstrated the great possibility of using K121 as a nonantibiotic agent for treatment of MRSA by evaluation of

Cobalt bis(1,2-dicarbollide) anion, namely, [3,3′-Co(1,2C2B9H11)2]− (Scheme 1), was first reported by Hawthorne et al. in 1965.13 It is the most studied metallacarborane structure units owing to its extraordinary physicochemical properties, such as low cytotoxicity, good water solubility, and amphiphilic behavior.14−22 Due to these specific properties, this rigid threedimensional complex and its derivatives were found to exhibit excellent biomedical applications, such as live-cell imaging,23 3485


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(here we hypothesize that 1 day represents a passage), as evidenced from the doubling of the MIC value (MIC10/MIC0 = 2, MIC0 = 8 μg/mL). This was followed by a 4-fold increase in MIC value at passage 19 (Figure 2), corresponding to the

(1) its anti-MRSA activity and the inhibition of biofilm formation; (2) the capability to combat drug-resistance development; and (3) the in vitro biocompatibility.

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RESULTS AND DISCUSSION Synthesis and Structure of K121. K121 is a typical member of a series of cobalt bis(1,2-dicarbollide) alkoxy derivatives that we previously designed and synthesized.30 By the reaction of [8,8′-μ-I-3,3′-Co(1,2-C2B9H10)2] with ethanol for 20 h at 30 °C, an orange solid, 8-ethoxy-8′-iododerivative of cobalt bis(1,2-dicarbollide) (K121), was obtained (Scheme 1). The characterization (1H NMR, 13C NMR, 11B NMR) of K121 is shown in the Supporting Information (Figure S1−S3). Anti-MRSA Property of K121. It has been recently reported that cobalt bis(1,2-dicarbollide) derivatives showed potential antimicrobial properties.15,28,29 Herein, we focused on the potent anti-MRSA activity of K121, a bifunctional cobalt bis(1,2-dicarbollide) alkoxy derivative. A series of assays were performed to confirm the antimicrobial activity of K121 against MRSA. In order to visualize the bactericidal effect of K121 by the naked eye, an agar plate colony-counting assay for MRSA was first performed (Figure 1a,b). Compared with the untreated group, K121-treated group could significantly reduce the number of colonies and completely kill the MRSA at suitable concentrations, showing the excellent antimicrobial characteristics of K121 against MRSA. Interestingly, K121 seems to act specifically against MRSA, since it almost ineffective on other ESKAPE superbugs, including Grampositive vancomycin-resistant Enterococcus faecium, Gramnegative multidrug-resistant Escherichia coli and P. aeruginosa (Table S1 in the Supporting Information). The specific antiMRSA activity is perhaps due to the fact that the cell wall structure of MRSA is different from those of other drugresistant bacteria. Further, we investigated the anti-MRSA property of K121 at different concentrations using a broth-dilution method.31 Consistent with the above colony-counting results, a concentration-dependent anti-MRSA characteristic was observed (Figure 1c). K121 exhibited observable anti-MRSA effect and the MRSA was completely eradicated at a concentration as low as 8 μg/mL (i.e., MIC = 8 μg/mL). The results show that the anti-MRSA capability of K121 is better than several other reported organometallic compounds.32−35 In order to determine the changes in the anti-MRSA activity over the time, the killing kinetic assays were performed. The assays showed that the MRSA cells were completely and rapidly eradicated after treatment with K121 at 2× MIC. As illustrated in Figure 1d, K121 caused a significant reduction in the number of viable MRSA colonies within 10 min and eradicated all the MRSA cells in 30 min at 2× MIC concentration (i.e., 16 μg/ mL). Obviously, K121 possesses the fastest killing kinetics of MRSA compared to other metallacarboranes reported in the literature to date,9,12 indicating that it has a potential value to treat clinical MRSA infections. Development of Drug Resistance. The problem of bacterial resistance has become one of the biggest obstacles to bacterial infections treatment. To assess the potential emergence of bacterial resistance after repeated use of K121, MRSA bacteria were exposed multiple times to K121 at a sublethal dose (1/3 of MIC). The conventional antibiotic vancomycin was used as a control. Multiple treatments with vancomycin induced significant drug resistance at passage 10

Figure 2. Changes in MIC and corresponding photo (inset) of the K121 against MRSA upon multiple sublethal dose exposures. The “gold standard” vancomycin was used as a control.

emergence of less-susceptible strains. In fact, plenty of studies focused on vancomycin-resistant MRSA isolated from patients,36 and reduced levels of bacterial susceptibility to vancomycin were well-known.37,38 Encouragingly, multiple treatments with K121 did not induce any changes in MIC value during the whole course of 20 passages, suggesting the effective avoidance of drug resistance development. Inhibition of Biofilm Formation. Bacterial biofilm, which is a complex architecture consisting of bacterial cells, protein, polysaccharide, and DNA, is a source of various recalcitrant and chronic infections and is notoriously difficult to eradicate.39 Under antibiotic pressure, for instance, the persistence and drug resistance mutation of staphylococcal infections is primarily due to biofilm formation.40,41 To assess its potency for biofilm formation inhibition, the inhibition ratio of MRSA biofilm were measured in the presence of different concentrations of K121 (Figure 3a). Obviously, K121 has excellent antibiofilm activity against MRSA under the experimental concentrations. At a quarter of MIC concentration (i.e., 2 μg/mL), K121 showed an inhibition ratio exceeding 80% in biofilm formation of MRSA. When the concentration reached 0.5× MIC (4 μg/mL), the inhibition ratio of the novel cobalt bis(1,2-dicarbollide) alkoxy derivative for the pathogen biofilm formation was higher than 97%. Furthermore, to provide a visual surface coverage of MRSA biofilm, the confocal laser scanning microscopy (CLSM) was carried out in the presence of subdouble MIC (4 μg/mL) of K121. CLSM images showed that the MRSA formed thick biofilms when grown in the absence of K121. In contrast, K121 at a concentration of 4 μg/mL could cause thinner and almost no cell clumps compared to those on normal biofilm architecture (Figure 3b). The stack 3-dimensional images showed that, in the control group, the average thickness of biofilm formed by MRSA was 10.36 μm. While MRSA was treated with 4 μg/mL of K121, the thickness dropped to 3.64 μm, proving the significant reduction in biofilm. Preliminary Antimicrobial Mechanism. In our previous study, we found that the antimicrobial mechanism of metallacarboranes mainly damages the bacterial cell wall/ 3486


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nanoparticles,46 selenium nanoparticles,47 are mainly related to ROS production. The action mechanisms of antibacterial through ROS generation have also been found in organometallic compounds, such as ferrocene-substituted heterotriorganometallic.48 By measuring the ROS levels using a commercial assay kit, our results demonstrated that the K121 significantly elevated ROS production inside MRSA (Figure 4c). In a negative control, we did not observe any increase in the ROS production on the water-treated group. The incorporation of antioxidant N-acetyl-L-cysteine (NAC) in the treatment group could eliminate the K121-induced ROS production, pulling down the ROS close to the initial level (Figure 4c), effectively inhibiting the MRSA cell membrane damage (Figure S4 in the Supporting Information). All these results revealed that K121 can effectively kill MRSA by increasing the ROS production and then inducing cell wall/ membrane damage, which ultimately leads to the death of MRSA. This might be helpful to explain why K121 could effectively combat drug resistance given the ability of bacteria to evolve and develop tolerance against membrane-disrupting antimicrobial agents.49 Thus, K121 should be able to serve as a promising nonantibiotic therapeutic agent against MRSA infections. Biocompatibility of K121. For clinical applications, it is mandatory to confirm the excellent biocompatibility toward mammalian cells by antimicrobial agent. To preliminary evaluate the in vitro cytotoxicity of K121, MTT assays were performed by using normal human liver cells (L02) as mammalian cell models. K121 showed negligible or no cytotoxicity to L02 cells even up to 64 μg/mL (Figure S5a in the Supporting Information), which is significantly higher than the MIC for MRSA (8 μg/mL). Meanwhile, the hemolytic behavior, a widely used evaluation index of selectivity of antibacterial agents over mammalian cells, was evaluated by treatment of human red blood cells (donated from a healthy male volunteer) with K121. Similar to the cytotoxicity results, as shown in Figure S5b in the Supporting Information, negligible hemolysis was observed for K121 at a relatively higher concentration (4× MIC). These results indicate that K121 has an excellent biocompatibility when it exerts its antiMRSA effect at MIC and can meet the need for further clinical use.

Figure 3. MRSA biofilm formation was inhibited by K121. (a) Inhibition ratio of MRSA biofilm in the presence of different concentrations of K121. (b) CLSM image of biofilm formation by MRSA in the absence of (left) and the presence of (right) K121 at 0.5× MIC concentration. The image shows the reconstructed 3dimensional biofilm images. Biofilm were stained with acridine orange, a widely used fluorescent biofilm biomass indicator. Scale bars = 25 μm.

membrane structure.9 Therefore, it is possible that K121 could also effectively damage the MRSA cell wall/membrane. On the basis of these considerations, we performed SYTO 9/ propidium iodide (PI) dyes costaining experiments in order to examine whether the integrity of MRSA cell membrane can be disrupted by K121 (Figure 4a). It is already known that the SYTO 9 dye could label all bacteria, intact and damaged membranes (green),42 whereas PI dye can only penetrates bacteria with damaged membranes, thus arousing a concomitant red fluorescence enhancement, causing a reduction in the SYTO 9 stain fluorescence when both dyes are present.42 The CLSM results showed that untreated (control group) MRSA cells nearly displayed only green fluorescence as the percentage of red fluorescent components was only 3.7%, as quantified by fluorometer. However, MRSA treated with K121 emitted much stronger red fluorescence, with 91.9% for the relevant percentage of red fluorescent components. These results demonstrated that K121-treated MRSA powerfully damaged cell membranes, thus allowing the internalization of PI dye into the cells. To further confirm the anti-MRSA mechanism of K121, scanning electron microscopy (SEM) was carried out for MRSA bacteria. According to the SEM micrographs presented in Figure 4b, the untreated MRSA cell showed smooth and intact globular morphology. After incubation with MIC of K121 for 2 h, the MRSA cell wall was sunken and damaged, which was consistent with our previous findings.9 It is evident that K121 strongly interacted with the MRSA cell wall and readily killed it by cell disruption. Furthermore, the reason behind the bacterial cell membrane damage, which might be mainly due to the rise of reactive oxygen species (ROS) induced by K121 treatment, was further exploited in this study. It has been reported that the killing mechanism of many antimicrobial agents, such as silver nanoclusters,43,44 gold nanoparticles,45 vanadium pentoxide

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CONCLUSION In the present study, we have successfully demonstrated a highly potent anti-MRSA agent, K121, a typical cobalt bis(1,2dicarbollide) alkoxy derivative. The specific antibacterial and biofilm inhibiting characteristics of K121 against MRSA have been first observed. In particular, a great killing efficiency that eradicated all MRSA cells within 30 min was achieved. K121 can effectively kill MRSA by increasing the ROS production, consequentially inducing cell wall/membrane damage, and ultimately leading to the death of MRSA. Interestingly, unlike conventional antibiotic vancomycin, repeated use of K121 did not induce drug resistance. The negligible cytotoxicity and hemolysis activity showed the excellent biocompatibility of K121, making it a potential candidate for treatment of persistent infections caused by MRSA.

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EXPERIMENTAL SECTION

Synthesis and Characterization of K121. The synthesis of K121 was described in our previous study.30 Briefly, a solution of precursor compound [8,8′-μ-I-3,3′-Co(1,2-C2B9H10)2, Scheme 1] (0.40 g) in 40 3487


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Figure 4. (a) CLSM images and corresponding statistical histograms of fluorescence intensity of MRSA without the treatment (control, watertreated) and with the treatment of K121. The dead cells were visualized by PI staining (red), while the SYTO 9 (green) helped to identify all cells. Scale bar is 50 μm. (b) SEM micrographs of MRSA without the treatment and with the treatment of K121. (c) Relative ROS yield by normalizing the ROS level after incubation for 2 h, where the ROS values of the control group was defined as one. 37 °C for 24 h in incubator. The viable colony counts were determined at different incubation times. The test was repeated in independent settings three times. Drug Resistance Study. Drug resistance was induced by repeatedly treating MRSA with K121 at sublethal doses.50 Briefly, the MICs of K121 and vancomycin (control) against MRSA was determined through 20 passages (here we hypothesize that a day represent a passage) of growth. MIC was measured by using the method described above. MRSA pressured to sub-MIC concentrations (1/3 of MIC at that particular passage) were allowed to regrow and reach a logarithmic growth phase before being used for MIC measurement of the subsequent passage. Development of drug resistance in MRSA was evaluated by recording changes in the MIC normalized to that of the first cell passage. Bacterial Biofilm Assay. Biofilm formation could be readily realized by using 96-well microtiter plate.51 MRSA were grown in LB for 12 h, diluted to 1.0 × 105 CFU/mL in the absence or presence of K121 at different concentrations. Aliquots of 100 μL were transferred to a sterile 96-well microtiter plate (5 wells per sample) and incubated at 37 °C for 24 h. After incubation, the suspension was discarded, and the plate was gently rinsed 3 times with sterile phosphate buffer saline (PBS), air-dried, and dyed with 200 μL of 1% (w/v) crystal violet per well for 10 min. After rinsing the plate for 3 times with ultrapure water, the dye associated with attached biofilm cells was dissolved with 200 μL of ethanol. The A595 of dye-ethanol solution was measured at 595 nm using a microplate reader (MK3, ThermoFisher). CLSM Observation of Biofilm. CLSM assay was carried out according to a previously reported method.9 MRSA incubated in LB medium was diluted to 1.0 × 105 CFU/mL after overnight culture,

mL of ethanol was rapidly shaken for 20 h. The solid was dissolved in 10 mL of acetone after evaporation of ethanol and mixed with an excess of cesium chloride solution. The mass spectra were recorded on a micro-OTOF II. NMR spectra (1H, 11B, 13C) were measured using a spectrometer (Bruker Avance-400). Anti-MRSA Activity. The K121 was dissolved in DMSO (below 0.5%) and diluted with sterile ultrapure water to the experimental concentration. Vancomycin was employed as a positive control to measure the antimicrobial property. MRSA strain was provided by the Yunnan Institute of Microbiology, Yunnan University, China. The strain was maintained on Luria−Bertani (LB) agar (tryptone 1 g, yeast extract 0.5 g, NaCl 1 g, agar 1.5 g, H2O 100 mL, pH = 7.2). An aliquot of 10 or 100 μg/mL K121 solutions were added to MRSA suspensions (∼1.0 × 108 CFU/mL), respectively. After incubation for 2 h, 100 μL of the appropriate diluted suspensions was coated onto the LB plate. After incubation for 24 h, the number of visible MRSA colonies was counted. The MIC was determined using a broth dilution method.31 Logarithmic-phase MRSA (∼1.0 × 106 CFU/mL) was inoculated into LB medium of 2 mL supplemented with the K121. The final concentrations range from 0.5 to 128 μg/mL. The as-prepared bacterial solutions were inoculated at 37 °C for 24 h. The viable colony counts were determined before and after incubation. The MIC is defined as the minimal concentration that inhibits visible colonies of tested strains as observed with the naked eye. Bactericidal Kinetic Assay. In order to assess the changes in the antimicrobial agent’s activity over the time, the killing kinetic tests have been carried out.12 Briefly, MRSA in the logarithmic growth phase (∼1.0 × 105 CFU/mL) was incubated with 2× MIC of K121 at 3488


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Organometallics subsequently transferred into sterile 6-well plate (preloaded with coverslips), and incubated at 37 °C for 24 h. After incubation, coverslips were gently rinsed 3 times with sterile PBS to rinse off the nonadherent cells, air-dried, and dyed with the 0.01% acridine orange at 4 °C for 20 min. Stained coverslips were gently rinsed 2 times with sterile PBS and observed with a CLSM (Nikon, Japan). Membrane Integrity Test. The bacterial membrane integrity test was determined using a commercial bacterial viability kit (Invitrogen). The kit provides a two-color fluorescence assay of bacterial viability, able to distinguish between intact membrane and damaged membrane. MRSA cells (∼1.0 × 108 CFU/mL) were treated with K121 in LB at 37 °C for 2 h. Then, the solution was stained with SYTO 9 and PI and incubated for 30 min. After that, the suspension was centrifuged, washed with ultrapure water, and resuspended in ultrapure water to the initial volume (1 mL). The sample was observed using a CLSM. The fluorescence intensity of the sample was registered with a fluorometer (RF-5301 PC, Shimadzu). ROS Generation. The ROS level was measured using a commercial ROS assay kit under the guidance of the manufacturer’s instructions. Bacteria cells (∼1.0 × 108 CFU/mL) were treated with K121 for 2 h at 37 °C. The bacterial sample was mixed with 10 μM 2′,7′-dichlorofluorescein diacetate (DCF-DA) and incubated for 1 h at room temperature in the dark. The fluorescence intensity of solutions was recorded with a fluorometer (RF-5301 PC, Shimadzu) using 488/ 525 nm as excitation/emission wavelengths. The relative ROS yield was calculated by normalizing the ROS level from the K121-treated group with the yield on the control water-treated group. As a validation experiment, 20 mM N-acetyl-L-cysteine (NAC), an antioxidant, was added along with K121, and the mixture solution was incubated for 2 h at 37 °C, keeping the same other steps. Bacterial Morphological Characterization. The morphological change of bacteria after treatment with K121 was observed by using a scanning electron microscope (SEM, Zeiss, Germany). Before imaging, the bacterial suspension was centrifuged and then immobilized with 2.5% glutaraldehyde for 1.5 h, dehydrating with graded ethanol (30%, 50%, 70%, 90%, and 100%, respectively). Cytotoxicity Assay. The MTT assay has been employed to preliminary evaluate the cytotoxicity of K121.52 Human embryo liver cells (L02) were seeded in a sterile 96-well microtiter plate 8 h and subsequently treated with different concentrations of K121 in medium for 24 h at 37 °C (5% CO2). Afterward, 20 μL of MTT (5 mg/mL) was separately added to wells and further incubated for 4 h. Thereafter, the cell supernatant was discarded and 150 μL of DMSO was added, with gentle shaking in the shaker for 10 min. The A490 of solutions was measured at 490 nm. Cell viability (%) was expressed as Atest/Acontrol × 100%, where A represents the absorbance at 490 nm. Hemolysis Assay. The red blood cells were obtained from human blood by centrifugation, gently washed with PBS and subsequently resuspended using PBS to prepare erythocyte suspension with a hematocrit of 2%. Then, different concentrations of K121 were incubated with isovolumetric 2% erythrocyte suspension at 37 °C for 2 h. Triton X-100 (1%) and PBS were employed as controls, respectively. After incubation, the supernatant was obtained by centrifugation and transferred to a 96-well microtiter plate (5 wells per treatment). The absorbance at 450 (A450) of supernatant was measured using a microplate reader. The hemolysis percentage was calculated as follows:

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AUTHOR INFORMATION

Corresponding Author

*E-mail for X.W.: [email protected]. ORCID

Hong Yan: 0000-0003-3993-0013 Xuemei Wang: 0000-0001-6882-7774 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National High Technology Research & Development Program of China (2015AA020502) and the National Natural Science Foundation of China (81325011 and 21175020) and the China-Russia Joint Grant (RFBR 16-53-53079, 21611130027) and the Russian Foundation for Basic Research Grant (RFBR 16-03-00724).

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hemolysis (%) = (A test − A negative control ) /(A positive control − A negative control ) × 100% where A represents the mean of absorbance value at 450 nm.

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Characterization and in vitro antibacterial activity of K121 and CLSM images of MRSA cells after treatment in conjunction with NAC, and biocompatibility evaluation (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.7b00426. 3489


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