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Tracking Reactions of Asymmetric Organo‐Osmium Transfer Hydrogenation Catalysts in Cancer Cells  by ICP-MS and nanofocussed x-ray fluorescence

Tracking Reactions of Asymmetric Organo‐Osmium Transfer Hydrogenation Catalysts in Cancer Cells  by ICP-MS and nanofocussed x-ray fluorescence

Session

Stream 4: Diamond Light Source Session 2

Authors

Dr Elizabeth Bolitho (3, 2), Dr James Coverdale (3), Dr Hannah Bridgewater (3), Dr Guy Clarkson (3), Dr Paul Quinn (2), Dr Carlos Sanchez-Cano (1), Professor Peter Sadler (3)

Affiliations

1. Center for Cooperative Research in Biomaterials (CIC biomaGUNE)
2. Diamond Light Source
3. University of Warwick

Keywords

Anticancer catalysts, bioorganometallics chemistry, X-ray fluorescence, organo-osmium complexes, transfer hydrogenation

Abstract text

[Os^II[(η⁶‐p‐cymene)(1, RR/SS‐MePh‐DPEN)] (MePh‐DPEN=tosyl‐diphenylethylene‐diamine) is a chiral 16‐electron organo‐osmium(II) half‐sandwich complex structurally derived from the well‐established Noyori Ru^II catalysts,¹ which shows high enantioselectivity and conversion rates. For example, reduction of acetophenone is 3‐fold more efficient (in turnover frequency, TOF) and more stable (normal atmospheric conditions) than its industrially‐used Ru^II analogue. ¹ Furthermore, once inside cells, and in presence of the non‐toxic hydride donor formate, 1 catalyses the enantioselective reduction of pyruvate, an essential precursor in cell metabolism, to natural L‐lactate or unnatural D‐lactate, depending on the chirality of the catalyst.² It can be assumed that the ability of such catalysts to cause metabolic perturbations in cells requires the presence of intact catalyst, which contributes to the antiproliferative activity and selectivity of 1 towards a variety of cancer cell lines.^2,3 Yet, its intracellular catalytic activity is most likely marked by low turnover numbers,² which might suggest some degradation of the complex inside cells. This is a common problem for synthetic metal catalysts, such as organometallic complexes designed to work under well‐defined chemical conditions, including inert atmospheres and in organic solvents. In order to optimise the design of synthetic intracellular catalysts, and increase their in cellulo catalytic and biological efficiency, it is important to investigate their fate in cells.

Previous work using ICP‐MS experiments on fractionated cancer cells treated with 1, showed 47 % cytosolic accumulation of the Os, suggesting that catalysis may take place in the cytosol.² Additionally, ca. 48 % of intracellular Os was present in the membrane/ particulate fraction (which contains organelles and membrane proteins), which may also implicate organelles (i.e. mitochondria or lysosomes) as cellular targets.² However, such studies did not provide information on the intracellular stability of the complex. To probe this, we have incorporated a bromine on a sulfonylphenyl substituent in the chelated Ph‐DPEN ligand, so generating [Os^II(η⁶‐p‐cym)(BrPh‐DPEN)] (2). A combination of nanofocussed synchrotron X‐ray fluorescence (XRF) and ICP‐MS allows not only osmium but also the chelated PhDPEN ligand to be tracked in cells using the bromine label.

First, pellets of A549 cancer cells treated with 2 under different conditions were digested at 353 K using TMAH and analysed by ICP-MS. Cellular accumulation of Os and Br in cells treated with 2 was determined under varying conditions (temperature, methyl-β-cyclodextrin, verapamil, time and cell fractionation). In all cases, significantly higher concentrations of Br compared to Os were found (>10- fold) suggesting 2 has a limited stability in a cellular environment. The endocytic contribution to the accumulation of 2 was investigated using a known endocytotic inhibitor, which reduced the quantity of intracellular Os with increasing concentration of inhibitor. Interestingly, intracellular levels of Br were initially reduced at the lowest inhibitor concentration, but plateaued with increasing concentration, thus it may be hypothesized that caveolae endocytosis mediates the uptake of the intact complex, but once inside the cell is degraded to release the Br-labelled ligand, which is retained intracellularly for longer. Similarly to 1, the dependence on drug efflux via. pathways associated with P-glycoprotein (using verapamil, a PGP inhibitor) revealed that the efflux of the Os moiety is dependent on PGP. However, Br accumulation was unaffected by verapamil treatment, suggesting that the Br-labelled PhDPEN ligand is not removed from cells by PGP. The extent of cellular efflux of 2 was monitored using varying exposure times and recovery in complex-free media, revealing maximum intracellular levels of Os 3-6 h, whereas, Br reached a maximum after 24 h exposure with around 30× more Br vs. Os, indicative of severe complex instability. Cellular fractionation studies of cells treated with 2 revealed significantly more Br vs. Os (>10-fold), with the majority of Os and Br observed in the membrane and cytoskeletal fractions. This suggests that some of complex is intact in the cytoplasm, where in-cell catalysis likely occurs. Negligible quantities of Os were present in the nuclear fraction which may rule out a DNA-binding mechanism of action. Interestingly, 10-20% Br was present in the nuclear fraction, perhaps as unbound Br-labelled PhDPEN. It may be hypothesized that the Os complex may facilitate the delivery of the PhDPEN into the cell (verified by the lack of toxicity of BsDPEN, IC₅₀>150 µM), where it can then be released and enter the nucleus. Whether the presence of BsDPEN is contributing to the anticancer activity is unknown, and requires further investigation

A549 cells grown on silicon nitride membranes were treated with various concentrations of 2 for 24 h, before being cryo‐fixed and freeze‐dried for subsequent analysis under ambient conditions. Nanofocussed synchrotron XRF at the I14 Beamline (Diamond) confirmed that some of 2 is likely to remain intact as judged by the co-localization of Os and Br in the cytosol, with significantly higher levels of Br vs. Os. Interestingly, XRF revealed the co-localization of Os and Br in small, spherical compartments (ca. 0.6 µm²)⁴ in cells treated with higher doses of 2, which may imply lysosomal breakdown and complex efflux. Remarkably, Br/Os ratios were lower in those areas than in the rest of the cell, suggesting the presence of higher concentrations of intact complex. The endosomal / lysosomal breakdown was further probed using chloroquine (which de-acidifies lysosomes), revealing a significant increase in potency and catalytic activity, which was accompanied by an increase in Os accumulation. 

Overall, we probed the structure and spatial localisation of a brominated transfer hydrogenation catalyst inside cells with ICP‐MS and nanoscale synchrotron XRF mapping, combined with cellular uptake and mechanistic studies. These experiments showed that the catalyst was degraded in cancer cells, probably through transport into acidic lysosomes following by reaction with cellular thiols. The chelated Br‐ligand (or a Br‐fragment), but not Os, is translocated into the nucleus. Such reactions help to explain the low intracellular TON estimated for these catalysts. This work demonstrates the utility of halogen tags as probes for MS and X‐ray based techniques which elucidate reactions of organometallic anticancer catalysts in cells. 

References

[1]  J. P. C. Coverdale, C. Sanchez-Cano, G. J. Clarkson, R. Soni, M. Wills, P. J. Sadler, Chem. Eur. J. 2015, 21, 8043– 8046.

[2] J. P. C. Coverdale, I. Romero-Canelón, C. Sanchez-Cano, G. J. Clarkson, A. Habtemariam, M. Wills, P. J. Sadler, Nat. Chem. 2018, 10, 347– 354.

[3] J. P. C. Coverdale, H. E. Bridgewater, J. I. Song, N. A. Smith, N. P. E. Barry, I. Bagley, P. J. Sadler, I. Romero-Canelón, J. Med. Chem. 2018, 61, 9246– 9255.

[4] E. M. Bolitho, J. P. C. Coverdale, H. E. Bridgewater, G. J. Clarkson, P. D. Quinn, C. Sanchez-Cano, P. J. Sadler.