Identification of clusters from reactions of ruthenium arene anticancer complex with glutathione using nanoscale liquid chromatography fourier transform ion cyclotron mass spectrometry combined with 18O-labeling

Fuyi Wang 0 1 2 4 Stefan Weidt 0 1 4 Jingjing Xu 0 1 4 C. Logan Mackay 0 1 4 Pat R. R. Langridge-Smith 0 1 4 Peter J. Sadler 0 1 3 4 0 Address reprint requests to Professor P. J. Sadler, Department of Chemistry, University of Warwick , Coventry CV4 7AL, UK 1 Published online December 15, 2007 Received October 2, 2007 Revised December 4, 2007 Accepted December 5, 2007 2 Institute of Chemistry, Chinese Academy of Sciences , Beijing, China 3 Department of Chemistry, University of Warwick , Coventry, United Kingdom 4 School of Chemistry, University of Edinburgh , Edinburgh, United Kingdom Reactions of the anticancer complex [( 6-bip)Ru(en)Cl] (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione ( -l-Glu-l-Cys-Gly; GSH), the abundant intracellular thiol, in aqueous solution give rise to two ruthenium cluster complexes, which could not be identified by electrospray mass spectrometry (ESI-MS) using a quadrupole mass analyzer. Here we use Fourier transform ion cyclotron mass spectrometry (nanoLC-FT-ICR MS) to identify the clusters separated by nanoscale liquid chromatography as the tetranuclear complex [{( 6-bip)Ru(GSO2)}4]2 (2) and dinuclear complex [{( 6-bip)Ru(GSO2)2}2]8 (3) containing glutathione sulfinate (GSO2) ligands. Use of 18OH2 showed that oxygen from water can readily be incorporated into the oxidized glutathione ligands. These data illustrate the power of high-resolution MS for identifying highly charged multinuclear complexes and elucidating novel reaction pathways for metallodrugs, including ligand-based redox reactions. (J Am Soc Mass Spectrom 2008, 19, 544 -549) 2008 American Society for Mass Spectrometry - ruthenium arene anticancer complex [( 6-bip)Ru(en)Cl] (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione ( -l-Glu-lCys-Gly; GSH) in aqueous solution [3]. Glutathione is present in almost all cells at millimolar concentrations and can detoxify some transition-metal ions. In this work, we have applied nanoscale liquid chromatography-Fourier transform ion cyclotron mass spectrometry (nanoLC-FTICR MS) to identify these clusters and 18OH2 derivatization to determine whether oxygen from solvent becomes incorporated into oxidized glutathione found in the products. [( 6-bip)RuCl(en)][PF6] (1) was synthesized as described elsewhere [10, 11]. Glutathione (GSH, reduced) and disodium hydrogen phosphate were purchased from Sigma (Dorset, UK), sodium dihydrogen phosphate from Aldrich (Dorset, UK), trifluoroacetic acid (TFAH) from Acros (Geel, Belgium) (Andover, MA) 18O-labeled Ction-metal ruthenium are of medical interest. omplexes of the second row, group 8 transiTwo RuIII complexes are currently undergoing clinical trials as anti-cancer agents, and RuII arene complexes have shown promising activity in model cancer systems [1]. Ruthenium has seven isotopes, making the mass isotopic pattern of ion peaks of ruthenium-containing compounds characteristic, yet complicated [2 6]. In our previous work [2, 3, 79], mass spectra acquired by electrospray ionization (ESI-MS) equipped with a quadrupole mass analyzer allowed unambiguous assignment of singly charged ion peaks of mononuclear ruthenium arene complexes as well as their adducts with amino acids, peptides, and DNA. However, low-resolution ESI-Q MS was unable to identify two multinuclear ruthenium clusters, which arose from the reaction of the water (95% H218O) from Cambridge Isotope Laboratories Instrumentation Positive-ion electrospray ionization mass spectra were obtained with a Bruker APEX III FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a 9.4 tesla actively shielded superconducting magnet (Magnex, UK). The instrument was modified inhouse by the replacement of the glass capillary with a heated metal transfer capillary held at a temperature of 423 K and a potential of 50 V. Ions were accumulated in an RF-only hexapole ion storage region for 2 s (1.2 10 7 mbar), before being transferred into the infinity cell (1.5 10 11 mbar) using sidekick trapping. An UltiMate 3000 series system (Dionex, UK) with nanoflow splitter was coupled to the mass spectrometer using an TriVersa NanoMate (Advion, Ithaca, NY) with an electrospray potential of 1.7 kV. Mobile phase A: water (for LC-MS application, Fisher Chemicals) containing 2% CH3CN and 0.1% TFAH; mobile phase B: CH3CN (for LC-MS application, Fisher Chemicals, oughborough, UK) containing 20% water and 0.1% TFAH. The sample was trapped and washed for 3 min at 30 L min 1 on a -Precolumn (300 m 5 mm, 5 m, 100 ). The sample was eluted onto an analytical PepMap 100 column (75 m 15 cm, 3 m, 100 ) held in a column-oven at 303 K. The flow rate was 300 nL min 1, and gradient (Solvent B) was as follows: 0 to 30% until 23 min, 30 to 100% from 23 to 24 min, 100% from 24 to 27 min, 100 to 0% from 27 to 29 min, and reset to 0% until 30 min. All spectra were acquired using XMass 7.02 (Bruker Daltonics) with 512 k data points in the range 90 3000 m/z. Bruker Daltonics Data Analysis software was used for analysis and post processing. Results and Discussion In unbuffered solution (pH ca. 3) and at 310 K, the ruthenium arene anticancer complex [( 6-bip) Ru(en)Cl] (1) reacted with 10 mol equivalent glutathione ( -l-Glu-l-Cys-Gly, GSH) to give two di-ruthenium glutathione complexes as the main products (Figure S1, which can be found in the electronic version of this article), of which the di-ruthenium triply-S bridged product centered at 13.99 min has been identified previously by conventional LC-ESI MS and NMR [3]. The ESI-Q MS showed that the fraction centered at 17.25 min contains a di-ruthenium glutathione sulfenate/ sulfinate complex (Figures S1 and S2). However, the concentration of multinuclear ruthenium clusters in the fraction eluted from 10.93 to 12.24 min was too low to allow good ESI MS analysis (Figures S1 and S2). The fraction centered at 17.25 min was also collected and concentrated for ESI-Q MS and NMR experiments. NMR results (Figure S3) show that only one set of proton resonances is observed for the two biphenyl ligands, suggesting that the two ruthenium centers in this complex are equivalent. No resonances are observed for the protons of the chelated ethylenediamine(en) ligand, indicating that the en ligands have been displaced from ruthenium by oxidized GSH ligands (Figure S3). The NMR sample was diluted with 1:1 H2O/CH3CN and analyzed by ESI-Q MS with various cone voltages in an attempt to obtain further structural information. However, the low-resolution of the mass spectra did not allow unambiguous identification of the di-ruthenium product either, although several fragmented ions observed with a cone voltage of 50 V (Figure S4) appeared to correspond to the release of one and two sulfinate (GSO2) ligands from the parent di-ruthenium complexes. The di-ruthenium product was also detectable in the reaction mixture of complex 1 with GSH under argon and physiologically-relevant condit (...truncated)