ࡱ> rtvydfh{ }   %` bjbjٕ C-&>>>>>>>Rz/z/z/8/0tRF^F14"4445V'6$K6i\k\k\k\k\k\k\$Z_ha\q>_655_6_6\>>44^}O}O}O_6>4>4i\}O_6i\}O}O>>}O4:1 psz/;I\}OU[^0F^}ObN|b}Ob>}O _6_6}O_6_6_6_6_6\\Oj_6_6_6F^_6_6_6_6RRRV!$RRRV!RRR>>>>>> Degradation of bidentate coordinated platinum(II)-based DNA intercalators by reduced L-glutathione Sharon Kemp, Nial J. Wheate, Michelle J. Pisani and Janice R. Aldrich-Wright* School of Biomedical and Health Sciences University of Western Sydney Locked Bag 1797 Penrith South DC, 1797, NSW Australia * Please address editorial correspondence to Assoc. Prof. Janice R. Aldrich-Wright via e-mail: j.aldrich-wright@uws.edu.au or via facsimile: +61 2 4620 3025. Keywords: anticancer, platinum(II), 1,10-phenanthroline, 1,2-diaminocyclohexane, reduced L-glutathione, L-methionine, degradation, cytotoxicity. Abbreviations: CB[n], cucurbit[n]uril; dach, 1,2-diaminocyclohexane; ESI, electrospray ionisation mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance spectroscopy; watergate, water suppression by gradient tailored excitation. Abstract We have examined the interaction of 1 ([(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+), 2 ([(5-methyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+), 3 ([(5,6-dimethyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)platinum(II)]2+) and 4 ([(5,6-dimethyl-1,10-phenanthroline)(ethylenediamine)platinum(II)]2+), with reduced L-glutathione and L-methionine. Both thiols degrade all four complexes, mainly by displacing the ancillary ligand and forming a doubly bridged dinuclear complex. The degradation half-life of 1-4 with methionine is > 7 days indicating that these reactions are not biologically relevant. The rate of degradation by glutathione appears to be particularly important and shows an inverse correlation to cytotoxicity. The least active complex 4 (t1/2 glutathione: 20 h) degrades fastest, followed by 3 (31 h), 2 (40 h), and 1 (68 h). The major degradation product 5 ([bis-(-{reduced L-glutathione}bis{5,6-dimethyl-1,10-phenanthroline}bis{platinum(II)}]2+) displays no cytotoxicity and is excluded as the source of the anticancer activity. Once bound by glutathione these metal complexes do not then form coordinate bonds with guanosine. Partial encapsulation of the complexes within cucurbit[n]urils is able to stop the degradation process. Introduction Over the last 10 years we have developed a family of over 60 platinum(II)- and 1,10-phenanthroline-based DNA intercalators, some of which demonstrate enormous potential in the treatment of human cancers that show resistance to current chemotherapeutic agents. ADDIN EN.CITE  ADDIN EN.CITE.DATA 1-6 With these DNA intercalators we have shown that ancillary ligand chirality and phenanthroline functional group substitution play an important role in determining their cytotoxicity; methylation in the 5, or 5 and 6, positions on the phenanthroline ligand and the ancillary ligand in an S,S-conformation, particularly with the 1,2-diaminocyclohexane ligand, confer the highest cytotoxicity. ADDIN EN.CITE  ADDIN EN.CITE.DATA 1-6 Although it is believed that DNA is the ultimate target for these metal complexes, DNA binding experiments have shown no significant differences in the DNA adducts formed between many of the DNA intercalators. ADDIN EN.CITE Kemp200721692169216917Kemp, SharonWheate, Nial J.Buck, Damian P.Nikac, MaricaCollins, J. GrantAldrich-Wright, Janice R.The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalatorsJ. Inorg. Biochem.J. Inorg. Biochem.1049-105810172007DOI: 10.1016/j.inorgbio.2007.04.0094 This suggests that all DNA adducts formed by this family of metal complexes are similarly cytotoxic and/or that DNA binding may not be the sole mechanism that determines their cytotoxicity. ADDIN EN.CITE Kemp200721692169216917Kemp, SharonWheate, Nial J.Buck, Damian P.Nikac, MaricaCollins, J. GrantAldrich-Wright, Janice R.The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalatorsJ. Inorg. Biochem.J. Inorg. Biochem.1049-105810172007DOI: 10.1016/j.inorgbio.2007.04.0094 We have hypothesised that differing cellular uptake levels or differing intracellular drug transport may determine how much of each DNA intercalator reaches its target intact, thereby affecting cytotoxicity. ADDIN EN.CITE Kemp200721692169216917Kemp, SharonWheate, Nial J.Buck, Damian P.Nikac, MaricaCollins, J. GrantAldrich-Wright, Janice R.The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalatorsJ. Inorg. Biochem.J. Inorg. Biochem.1049-105810172007DOI: 10.1016/j.inorgbio.2007.04.0094 The tripeptide, reduced L-glutathione ((-glutamylcysteinylglycine, GSH, Figure 1), is abundant in cells at concentrations between 0.5-10 mM ADDIN EN.CITE Jung200722422242224217Jung, YongwonLippard, Stephen J.Direct cellular responses to platinum-induced DNA damageChem. Rev.Chem. Rev.1387-1407107520077 and is involved in many cellular processes, including: metabolism, protection of cells from oxidative stress and detoxification and/or bioactivation of drugs. ADDIN EN.CITE  ADDIN EN.CITE.DATA 8-10 L-methionine is an essential amino acid which contains a thioether group capable of binding to platinum atoms (Figure 1). The anticancer drugs cisplatin and BBR3464, ADDIN EN.CITE Wheate200355517Wheate, N. J.Collins, J. G.Multi-nuclear platinum complexes as anti-cancer drugsCoord. Chem. Rev.Coord. Chem. Rev.133-1452411-22003JunISI:000183738900007<Go to ISI>://000183738900007 11 the latter an agent capable of overcoming cisplatin resistance in vitro/in vivo, ADDIN EN.CITE Wheate200512121217Wheate, N. J.Collins, J. G.Multi-nuclear platinum drugs: A new paradigm in chemotherapyCurr. Med. Chem. - Anti-Cancer AgentsMulti-nuclear platinum drugsCurr. Med. Chem. - Anti-Cancer Agents267-279532005ReviewWheate200355517Wheate, N. J.Collins, J. G.Multi-nuclear platinum complexes as anti-cancer drugsCoord. Chem. Rev.Coord. Chem. Rev.133-1452411-22003JunISI:000183738900007<Go to ISI>://000183738900007 10, 11 are readily degraded into non-active complexes by both glutathione and methionine. ADDIN EN.CITE Wheate200512121217Wheate, N. J.Collins, J. G.Multi-nuclear platinum drugs: A new paradigm in chemotherapyCurr. Med. Chem. - Anti-Cancer AgentsMulti-nuclear platinum drugsCurr. Med. Chem. - Anti-Cancer Agents267-279532005ReviewWheate200355517Wheate, N. J.Collins, J. G.Multi-nuclear platinum complexes as anti-cancer drugsCoord. Chem. Rev.Coord. Chem. Rev.133-1452411-22003JunISI:000183738900007<Go to ISI>://000183738900007 10, 11 This degradation occurs through the ability of the sulphur atoms to displace both the chloride and monodentate am(m)ine ligands of cisplatin and BBR3464. This degradation leads to decreased levels of active drug reaching its cellular target, DNA, and modulation of the drugs inter- and intracellular transport. Importantly, glutathione and methionine are unable to displace bi- or tridentate amine ligands (like ethylenediamine and diethylenetriamine), which is why these ligands are routinely used in thiol protein degradation experiments. ADDIN EN.CITE Reedijk199917417417417Reedijk, J.Why does cisplatin reach guanine-N7 with competing S-donor ligands available in the cell?Chem. Rev.Chem. Rev.2499-25109991999SepISI:000082569800011<Go to ISI>://000082569800011 del Socorro Murdoch199923182318231817del Socorro Murdoch, PiedadKratochwil, Nicole A.Parkinson, John A.Patriarca, MarinaSadler, Peter J.A novel dinuclear diaminoplatinum(II) glutathione macrochelateAngew. Chem. Int. Ed.Angew. Chem. Int. Ed.2949-29513819199912, 13 Their inertness limits the number of degradation products formed and thus simplifies spectral analysis. ADDIN EN.CITE  ADDIN EN.CITE.DATA 14-16 To date there have been no papers describing the removal of bi- or tridentate amine ligands from platinum complexes by thiols or thioethers. In this study we report the unexpected degradation by reduced L-glutathione, and to a lesser extent the degradation by L-methionine, of four platinum(II)-based DNA intercalators, 1 ([(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+), 2 ([(5-methyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+), 3 ([(5,6-dimethyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)platinum(II)]2+) and 4 ([(5,6-dimethyl-1,10-phenanthroline)(ethylenediamine)platinum(II)]2+) (Figure 1). The degradation process and products were examined using 1H and 195Pt NMR, electrospray ionisation mass spectrometry and in vitro L1210 murine leukaemia growth inhibition assays. Results The platinum(II)-based DNA intercalator complexes 1-4 (1 mM) were reacted with 4 mM reduced L-glutathione (glutathione) or L-methionine (methionine) at 37 (C in either unbuffered D2O or phosphate buffered saline (pH 7) in D2O for periods of up to seven days. All four DNA intercalators are reactive towards glutathione and methionine as indicated by a colour change from either a clear pink (2), cream (3, 4) or pale-yellow (1) solution to a dark brown solution, with the formation after approximately three days of an orange precipitate, which increases in amount as the reaction proceeds. The precipitate is not soluble in aqueous solvents and shows only limited solubility in hot dimethylsulphoxide, which excluded its analysis by standard chemical instrumentation. The reaction of all four metal complexes with methionine is very slow (t1/2 > 7 days) indicating that the metal complexes reactions with this amino acid in vitro/in vivo are probably not relevant to the structure-activity relationship of this family of DNA intercalators. As such, there was no further examination of their reaction kinetics or products. Reactions with glutathione, however, appear to be particularly important. All four metal complexes react with glutathione at differing rates and with what appears to be an inverse correlation with their in vitro cytotoxicity in the L1210 murine leukaemia cell line (Paired students t-test, P < 0.05). The least active anticancer DNA intercalator, 4, reacts the fastest (t1/2, 20 h), followed by 3 (t1/2, 31 h), 2 (t1/2, 40 h), then 1 (t1/2, 68 h), in the order corresponding to their increasing cytotoxicity (Table 1). The reaction half-lifes of the metal complexes were not signficantly different whether straight D2O or phosphate buffered saline (pH 7) D2O was used, indicating that drug degradation is not due to a decrease in the pH of the solution as protons are lost from the thiol. 1H NMR analysis of the reduced L-glutathione"metal complex reaction solution. The reaction of all four metal complexes with glutathione and methionine is also observed by the appearance, in 1H NMR spectra, of new metal complex resonances in the aromatic region, and new glutathione resonances in the aliphatic region. As an indicative example, the 1H NMR spectra of 4 shows just two doublet resonances and one doublet of doublets resonance at the beginning of the reaction (Figure 2). After 24 h, another, non-symmetrical set of phenanthroline resonances (four doublets and two doublet of doublets resonances) is observed and can be assigned as the major degradation product. After 48 h a third set of non-symmetrical phenanthroline resonances are clearly resolvable, which are assigned as a minor degradation product. For all four metal complexes the aromatic resonances corresponding to free metal complex also move downfield as the reaction progresses. This result is consistent with a decrease in free metal complex concentration, as the chemical shift of intercalator resonances in aqueous solvents has been shown previously to change with varying intercalator concentration. ADDIN EN.CITE Kemp19221922192217Kemp, SharonWheate, Nial J.Wang, ShaoyuCollins, J. GrantRalph, Stephen F.Day, Anthony I.Higgins, Vincent J.Aldrich-Wright, Janice R.Encapsulation of platinum-based DNA intercalators within cucurbit[6,7,8]urilsJ. Biol. Inorg. Chem.J. Biol. Inorg. Chem.969-97912200717 The chemical shift varies due to - interactions between stacked intercalator molecules; as the concentration varies the number of - interactions changes, with greater molecular stacking inducing 1H downfield resonance shifts and fewer - interactions inducing upfield shifts. 195Pt NMR analysis of the reduced L-glutathione"metal complex reaction solutions. The 195Pt NMR of the reaction mixture of between 1 or 3 and glutathione reveals two resonances; one large peak at -2828 ppm, consistent with the unreacted N4 coordination sphere of 1 or 3, and another resonance slightly upfield at -2890 ppm, consistent with a dinuclear platinum degradation product bridged by two individual glutathione molecules. ADDIN EN.CITE Papadia200522402240224017Papadia, ParideMargiotta, NicolaBergamo, AlbertaSava, GianniNatile, GiovanniPlatinum(II) complexes with antitumoral/antiviral aromatic heterocycles: Effect of glutathione upon in vitro cell growth inhibitionJ. Med. Chem.J. Med. Chem.3364-3371482005del Socorro Murdoch199923182318231817del Socorro Murdoch, PiedadKratochwil, Nicole A.Parkinson, John A.Patriarca, MarinaSadler, Peter J.A novel dinuclear diaminoplatinum(II) glutathione macrochelateAngew. Chem. Int. Ed.Angew. Chem. Int. Ed.2949-29513819199913, 14 In some spectra, a very small resonance is also observed at -3509 ppm (> 1%). Similar to 1, 4 also gives two resonances at -2828 and -2916 ppm, which are the free metal complex and a doubly bridged dinuclear complex, respectively. The 195Pt NMR spectrum of 2 however gives three distinct resonances (Figure 3). The first at -2828 ppm is consistent with unreacted 2 and the second resonance, at -2916 ppm, is again consistent with a doubly bridged dinuclear platinum degradation product. A third resonance appears (600 ppm upfield from free 2 at -3551 ppm and is consistent with a N3S coordination sphere. ADDIN EN.CITE Oehlsen200531431431417Oehlsen, M. E.Hegmans, A.Qu, Y.Farrell, N.A surprisingly stable macrochelate formed from the reaction of cis dinuclear platinum antitumor compounds with reduced glutathioneInorg. Chem.Inorg. Chem.3004-30064492005MayISI:000228813400008<Go to ISI>://000228813400008 Oehlsen200333433433417Oehlsen, M. E.Qu, Y.Farrell, N.Reaction of polynuclear platinum antitumor compounds with reduced glutathione studied by multinuclear (H-1, H-1-N-15 gradient heteronuclear single-quantum coherence, and Pt-195) NMR spectroscopyInorg. Chem.Inorg. Chem.5498-550642182003SepISI:000185219500015<Go to ISI>://000185219500015 18, 19 The degradation resonances of all four metal complexes increase in intensity as the reactions proceed, and can account for between 20 and 60% of the total platinum in the solutions, but are always comparable in relative size to the degradation resonances observed in the equivalent 1H NMR spectra. Electrospray ionisation mass spectrometry analysis of the reduced L-glutathione"metal complex reaction solution. The ESI+ mass spectra of all four metal complexes after reaction with glutathione gave ion peaks between 200-1400 m/z consistent with a number of platinum degradation products. As an indicative example, the ESI+ mass spectrum of 1 [M-H+]+ showed the free drug as an ion peak at 516.4 m/z. A major degradation product peak occurred at 709.5 m/z, consistent with a 2+ bisplatinum complex containing two phenanthroline ligands and two individual bridging glutathione ligands (Degradation product 5, Figure 4). This peak could also mask the peak of a mononuclear platinum degradation product where one glutathione molecule displaces a diaminocyclohexane (dach) ligand and binds the platinum atom through two coordination sites. Another major ion isotope peak occurs at 823.6 m/z which indicates the presence of a 1+ complex containing platinum, phenanthroline, diaminocyclohexane and glutathione, indicating that the dach is still attached, possibly via one amine group and the glutathione is bound through a single site, possibly through the sulphur group (Degradation product 6). A further minor ion peak at 615.5 m/z can be assigned to a degradation product where the phenathroline ligand has been removed and replaced by a single glutathione molecule (Degradation product 7). Synthesis of 5 ([bis-(-{reduced L-glutathione}bis{5,6-dimethyl-1,10-phenanthroline}bis{platinum(II)}]2+). The metal complex was synthesised by first reacting an equimolar concentration of K2PtCl4 and 5,6-dimethyl-1,10-phenanthroline in dimethylsulphoxide. The chloride ligands were removed from the resultant crystals by the addition of AgNO3 in dimethylformamide, before an equimolar amount of reduced L-glutathione was added. The desired product was purified by precipitation with diethyl ether then eluted on a C18-reverse phase column with a 50:50 mixture of acetonitrile and water. Metal complex 5 was characterised using 1H (one-dimensional Watergate and two-dimensional nuclear Overhauser effect spectroscopy and double quantum correlated spectroscopy) and 195Pt NMR, ESI+ mass spectrometry and elemental analysis. In the 1H NMR spectrum, there are two doublet resonances at 9.31 and 8.75 ppm assigned as the phenanthroline H2/H9 protons, the resonances at 8.96 and 8.66 ppm are assigned as the H4/H7 protons and two doublet of doublet resonances at 8.25 and 7.75 ppm are assigned as the H3/H8 protons (Figure 5). Two phenanthroline methyl singlet resonances appear in the aliphatic region at 2.72 and 2.64 ppm. There appears to be two sets of glutathione resonances which overlap giving the appearance of mutliplets. Finally, the glycine and cysteine amine resonances have shifted significantly in 5 compared to free reduced L-glutathione; in the Watergate 1H NMR spectrum of 5 the glycine NH resonance has shifted downfield from 8.42 to 8.62 ppm and the cysteine NH resonance has shifted upfield from 8.37 to 8.18 ppm. Normally, the phenanthroline ligand only has two doublet resonances and one doublet of doublet resonance in the aromatic region and only one methyl resonance in the aliphatic region. The 1H NMR spectrum of 5 therefore indicates that the metal complex is not symmetrical. Previously, Papadia et al. have described a dinuclear platinum(II) complex containing two phenanthroline ligands, which is bridged by two individual glutathione molecules binding through the sulphur groups (i.e. 5 but without the phenanthroline methyl groups). ADDIN EN.CITE Papadia200522402240224017Papadia, ParideMargiotta, NicolaBergamo, AlbertaSava, GianniNatile, GiovanniPlatinum(II) complexes with antitumoral/antiviral aromatic heterocycles: Effect of glutathione upon in vitro cell growth inhibitionJ. Med. Chem.J. Med. Chem.3364-337148200514 This metal complex can exist as two different isomers with the glycine residues either in a syn or anti position to each other (Figure 6). ADDIN EN.CITE Papadia200522402240224017Papadia, ParideMargiotta, NicolaBergamo, AlbertaSava, GianniNatile, GiovanniPlatinum(II) complexes with antitumoral/antiviral aromatic heterocycles: Effect of glutathione upon in vitro cell growth inhibitionJ. Med. Chem.J. Med. Chem.3364-337148200514 The 1H NMR spectrum of 5 is therefore consistent with a mixture of isomers of a doubly bridged dinuclear platinum(II) complex. The 195Pt NMR and ESI+ mass spectra are also consistent with the proposed chemical structure. A single 195Pt NMR resonance is observed at -2893 ppm, similar to that reported by Papadia et al. at -2871 ppm (see Figure 5). ADDIN EN.CITE Papadia200522402240224017Papadia, ParideMargiotta, NicolaBergamo, AlbertaSava, GianniNatile, GiovanniPlatinum(II) complexes with antitumoral/antiviral aromatic heterocycles: Effect of glutathione upon in vitro cell growth inhibitionJ. Med. Chem.J. Med. Chem.3364-337148200514 In the ESI+ mass spectrum, 5 shows a single major ion peak (100%) at 709.1 m/z. The isotopic pattern of the ion peak is consistent with the theoretical isotopic distribution for a doubly bridged dinuclear platinum complex (C48H55N10O12Pt2S2+, data not shown), which further confirms the proposed chemical structure of 5. Reaction of 56MEGL with 52 -guanosine monophosphate. 52 -Guanosine monophosphate (2 mM) and 5 (1 mM) were incubated at 37 oC in unbuffered D2O (600 (L) and the 1H NMR spectrum recorded at intervals for up to seven days. There was no change in either the line width or chemical shift of the guanosine H8 or H12 resonances nor the 5 proton resonances, indicating that the reduced L-gluthathione ligands are not displaced by guanosine. Reaction of the metal complexes with reduced L-glutathione in the presence of cucurbit[n]uril (CB[n]). The metal complexes 1-4 (1 mM) were incubated with CB[n] (n = 6, 7 or 8) (1 mM) and reduced L-glutathione (4 mM) in unbuffered D2O (600 (L) at 37 oC and 1H NMR spectra recorded. No metal complex degradation products were observed in any spectra at intervals of up to seven days. Discussion In this study we have examined the reaction of metal complexes 1-4 from a family of platinum(II)-based DNA intercalator complexes with reduced L-glutathione (glutathione) and L-methionine (methionine). These four metal complexes were chosen because they display cytotoxicities ranging from 1 which is up to 100-fold more active than cisplatin, to 4 which is less cytotoxic than cisplatin (see Table 1). Metal complex 1 is also considerably more active than cisplatin in several human cancer cells lines. As such, these four metal complexes allowed us to examine the structure-activity relationship of thiol degradation in this family of metal complexes. These metal complexes differ mostly in the structure and chirality of their ancillary ligands. Both 1 and 2 have 1S,2S-diaminocyclohexane as the ancillary ligand, 3 contains the same ligand but with different chirality (i.e. R,R) and 4 contains the achiral ligand, ethylenediamine. Unexpectedly, metal complexes 1-4 react with glutathione and to a lesser extent with L-methionine. This result is unexpected because bi- and tridentate amine ligands have previously been shown to be inert to removal by thiols and thiolates, regardless of the other coordinating ligands. ADDIN EN.CITE Reedijk199917417417417Reedijk, J.Why does cisplatin reach guanine-N7 with competing S-donor ligands available in the cell?Chem. Rev.Chem. Rev.2499-25109991999SepISI:000082569800011<Go to ISI>://000082569800011 12 This is in contrast to cisplatin and BBR3464, which contain only monodentate chloride and am(m)ine ligands and readily undergo subsitution reactions with both types of thiols. The reaction of the metal complexes with methionine does not appear to be biologically relevant as the half-lifes are greater than seven days. Their reaction with glutathione, however, appears to be particularly relevant and shows an inverse correlation to their cytotoxicity in the L1210 murine leukaemia cell line (see Table 1). The time difference in reaction half-lifes is also significant; 1 is more than 2-fold slower to react with glutathione than 4. The reaction of all four metal complexes with glutathione, however, is considerably slower than cisplatin or BBR3464 (6 to 20-fold compared to cisplatin). ADDIN EN.CITE Corden198722472247224717Corden, Brian J.Reaction of platinum(II) antitumor agents with sulfhydral compounds and the implications for nephrotoxicityInorg. Chim. ActaInorg. Chim. Acta125-130137198720 Despite the relatively long degradation half-lifes we conclude that the reaction of these DNA intercalators with glutathione is biologically relevant as platinum drugs have been shown to have long circulating life-times in the human body. In humans only half of the drug dose is excreted within nine days for oxaliplatin, ADDIN EN.CITE Hartmann200320102010201017Jorg Thomas HartmannHans-Peter LippToxicity of platinum compoundsExpert Opin. Pharmacother.Expert Opin. Pharmacother.889-90146200321 5.8 days for carboplatin, ADDIN EN.CITE Lokich199822412241224117Lokich, J.Anderson, N.Carboplatin versus cisplatin in solid tumors: An analysis of the literature.Ann. Oncol.Ann. Oncol.13-219199822 and 5.4 days for cisplatin. ADDIN EN.CITE Lokich199822412241224117Lokich, J.Anderson, N.Carboplatin versus cisplatin in solid tumors: An analysis of the literature.Ann. Oncol.Ann. Oncol.13-219199822 A reliable human in vivo half-life of BBR3464 is yet to be reported but a recent Phase I clinical trial found that BBR3464 was still being excreted 15 days after treatment. ADDIN EN.CITE Sessa200045445445417Sessa, C.Capri, G.Gianni, L.Peccatori, F.Grasselli, G.Bauer, J.Zucchetti, M.Vigano, L.Gatti, A.Minoia, C. Liati, P.Van den Bosch, S.Bernareggi, A.Camboni, G.Marsoni, S.Clinical and pharmacological phase I study with accelerated titration design of a daily times five schedule of BBR3464, a novel cationic triplatinum complexAnn. Oncol.Ann. Oncol.977-98311200023 It is unclear why these DNA intercalators degrade at differing rates. It is possible that the chirality of 3, or lack of chirality in the case of 4, allows a chiral-specific interaction with one or more amino acids of the glutathione, whereas for 1 and 2 the interaction may be less favourable. Such an interaction may stabilise a 3 or 4 complex that is longer-lived than the 1- or 2-glutathione interactions, or, the chirality of 1 or 2 may provide a steric bulk that prevents glutathione from more closely approaching the platinum atom. Certainly steric factors do play a role in sulphur attack on platinum centres, and one drug currently undergoing clinical trials, cis-[PtCl2(2-methyl-pyridine)NH3] (picoplatin) was designed specifically so that the methyl group sits directly over the platinum atom, providing steric hinderance to sulphur attack. ADDIN EN.CITE Wong199913131317Wong, E.Giandomenico, C. M.Current status of platinum-based antitumor drugsChem. Rev.Chem. Rev.2451-24669991999SepISI:000082569800009<Go to ISI>://000082569800009 24 Unfortunately no chiral-specific interactions between the free ligands S,S- or R,R-dach and glutathione are observed. At concentrations of 4 mM there are no significant changes in the 1H NMR proton resonances of either the diaminocyclohexane compounds or glutathione when combined. There are no intermolecular cross-peaks in two-dimensional rotating frame Overhauser effect NMR spectra. Examination of the reaction products of the metal complexes with glutathione using 1H and 195Pt NMR and mass spectrometry reveal the formation of a number of degradation products. For all four metal complexes the major degradation product appears to be a dinuclear platinum complex with two phenanthroline ligands and two individual glutathione molecules coordinated to the platinum atoms through their sulphur groups forming a doubly bridged complex, 5 ([bis-(-{reduced L-glutathione}bis{5,6-dimethyl-1,10-phenanthroline}bis{platinum(II)}]2+) (see Figure 4). Formation of a doubly bridged dinuclear platinum complex is consistent with the product observed by del Socorro Murdoch et al. from the reaction of [Pt(en)Cl2] with reduced glutathione. ADDIN EN.CITE del Socorro Murdoch199923182318231817del Socorro Murdoch, PiedadKratochwil, Nicole A.Parkinson, John A.Patriarca, MarinaSadler, Peter J.A novel dinuclear diaminoplatinum(II) glutathione macrochelateAngew. Chem. Int. Ed.Angew. Chem. Int. Ed.2949-29513819199913 Mass spectrometry also revealed the formation of other minor degradation species. From both the 1H and 195Pt NMR and ESI+ mass spectra we tentatively assigned the most relevant minor product as a mononuclear species where the ancillary ligand has either been fully displaced by a single glutathione molecule which is bound through the cysteine sulphur and nitrogen atoms, or where the ancillary ligand has been partially displaced by a glutathione molecule which is bound solely through its sulphur group (i.e. Degradation product 6 in figure 4). Both minor degradation products would be expected to have non-symmetrical phenanthroline resonances and an N3S coordination sphere yielding a 195Pt NMR resonance of around 3500 ppm, consistent with the resonance observed at -3550 ppm in Figure 3. Whilst both of these observed metal complexes may be minor degradation products in their own right, both may also simply be intermediates in the formation of 5. To further examine the major degradation product, 5 was synthesised and purified separately. Metal complex 5 has 1H and 195Pt NMR spectra identical to the major degradation product of 1 and an ESI+ mass spectrum showing a single peak with isotope patterning consistent with a C48H55N10O12Pt2S2+ (i.e. [5]-H+]+) ion. It is possible that much of the cytotoxicity of this family of DNA intercalators is derived not from the metal complexes themselves, but from their degradation products. To test this hypothesis, 5 was examined for cytotoxicity in the L1210 murine leukaemia cell line. The metal complex 5 displays no growth inhibition at concentrations up to 50 (M. Also of significant interest is the ability of thiols to act as reservoirs for platinum(II)-based drugs inside the cell. Cisplatin is easily degraded by both glutathione and methionine, but when degraded by glutathione can still go on to form coordinate bonds at the N7 position of guanosine. ADDIN EN.CITE Reedijk199917417417417Reedijk, J.Why does cisplatin reach guanine-N7 with competing S-donor ligands available in the cell?Chem. Rev.Chem. Rev.2499-25109991999SepISI:000082569800011<Go to ISI>://000082569800011 12 It was therefore of interest to determine whether the anticancer activity of this family of metal complexes arises not from DNA intercalation but from a cisplatin-like coordinate adduct with DNA formed from their glutathione degradation products. To test this hypothesis 5 was incubated with 52 -guanosine monophosphate for time periods of up to seven days and the 1H NMR spectrum of the solution recorded at intervals. At no stage was there evidence of coordinate adduct formation to guanosine (as would have been indicated by downfield shifts of the guanosine H8 and H12 proton resonances), suggesting that these metal complexes do not act by forming coordinate covalent adducts with DNA through their degradation products. The glutathione reaction half-lifes of the four metal complexes, taken together with the cytotoxicity and guanosine binding results of metal complex 5 suggest two things: (a) the major degradation product of this family DNA intercalators is not the cause of their cytotoxicity, and (b) if degradation by glutathione can be slowed, or even prevented, then the metal complexes cytotoxicity and efficacy can be further increased. Previously, we have examined the use of cucurbit[n]urils (CB[n], where n = 5, 6, 7, 8 or 10) as drug delivery vehicles (Figure 7). ADDIN EN.CITE  ADDIN EN.CITE.DATA 5, 17, 25, 26 CB[6], CB[7] and CB[8] were generally found to bind over the metal complexes ancillary ligands (diaminocyclohexane or ethylenediamine) with a variety of platinum(II)-based DNA intercalators. ADDIN EN.CITE Wheate200722382238223817Wheate, Nial J.Taleb, Robin I.Krause-Heuer, Anwen M.Cook, Rebekah L.Wang, ShaoyuHiggins, Vincent J.Aldrich-Wright, Janice R.Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehiclesDalton Trans.Dalton Trans.5055-5064200710.1039/b704973kKemp19221922192217Kemp, SharonWheate, Nial J.Wang, ShaoyuCollins, J. GrantRalph, Stephen F.Day, Anthony I.Higgins, Vincent J.Aldrich-Wright, Janice R.Encapsulation of platinum-based DNA intercalators within cucurbit[6,7,8]urilsJ. Biol. Inorg. Chem.J. Biol. Inorg. Chem.969-9791220075, 17 This encapsulation was found to modulate the cytotoxicity of the metal complexes, with CB[6] increasing the cytotoxicity of some of the DNA intercalators. Given these results, we were interested to see if CB[n] could prevent metal complex degradation by glutathione. When all four metal complexes were incubated with one mole equivalent of CB[6], CB[7] or CB[8] and four mole equivalent of glutathione, no degradation was observed in the 1H NMR spectra for time periods up to seven days. This result is consistent with similar experiments where CB[n] was able to reduce the rate of reaction of mono- and multinuclear platinum(II)-based anticancer complexes with biological nucleophiles (e.g. L-cysteine and guanosine). ADDIN EN.CITE  ADDIN EN.CITE.DATA 25, 27, 28 We are now examining CB[n] derivatives, different macrocycles and other molecular structures as drug delivery vehicles for not just this family of platinum(II)-based DNA intercalators but also for other platinum(II)-based anticancer complexes that are of interest. ADDIN EN.CITE  ADDIN EN.CITE.DATA 5, 29, 30 Conclusions This family of platinum(II)-based DNA intercalator complexes is unexpectedly degraded by reduced L-glutathione, and to a lesser extent by L-methionine, where the loss of the ancillary (either ethylenediamine or 1,2-diaminocyclohexane) or intercalating ligand (5-methyl-1,10-phenanthroline or 5,6-dimethy-1,10-phenanthroline) forms a mixture of stereoisomer mono- and dinuclear reaction products. The major degradation product is a dinuclear platinum complex with two phenanthroline ligands linked through the sulphur atoms of two individual reduced L-glutathione molecules. While the degradation of the DNA intercalators by methionine does not appear to be biologically relevant (half-lifes > 7 days), degradation by glutathione occurs in a metal complex specific manner. This degradation appears to be inversely correlated with the cytotoxicity of the platinum complexes, where the most active complexes degrade at the slowest rate and the less active complexes degrade at the fastest rate. Synthesis and cytotoxicity of the proposed major degradation product, [bis-(-{reduced L-glutathione}bis{5,6-dimethyl-1,10-phenanthroline}bis{platinum(II)}]2+ (5) demonstrated no cytotoxicity up to 50 (M, and thus is not the cause of the cytotoxicity of this family of platinum(II)-based DNA intercalators. The results therefore indicate that if degradation of these platinum(II)-based DNA intercalators can be further slowed or even stopped, then the cytotoxicity and efficacy of the already highly active anticancer DNA intercalator complexes, like 1 and 2, could be further increased. Experimental Section Materials. The platinum(II) complexes 1-4 were synthesised as previously described. ADDIN EN.CITE Wheate200722382238223817Wheate, Nial J.Taleb, Robin I.Krause-Heuer, Anwen M.Cook, Rebekah L.Wang, ShaoyuHiggins, Vincent J.Aldrich-Wright, Janice R.Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehiclesDalton Trans.Dalton Trans.5055-5064200710.1039/b704973k5 Cucurbit[6, 7 and 8]urils were provided by Dr Anthony Day, UNSW@ADFA and made as previously described. ADDIN EN.CITE Day200134343417Day, A.Arnold, A. P.Blanch, R. J.Snushall, B.Controlling factors in the synthesis of cucurbituril and its homologuesJ. Org. Chem.J. Org. Chem.8094-810066242001NovISI:000172489500023<Go to ISI>://000172489500023 31 L-methionine was purchased from Sigma. Reduced L-glutathione, 5,6-dimethyl-1,10-phenanthroline, 5-methyl-1,10-phenanthroline, ethylenediamine, 1R,2R-diaminocyclohexane and 52 -guanosine monophosphate were purchased from Sigma-Aldrich. 1S,2S-diaminocyclohexane was purchased from Fluka. AgNO3 was purchased from BDH Chemicals. Deuterated solvents D2O (99.9%) and d7-DMF (99.9%) were purchased from Cambridge Isotope Laboratories. All general solvents were used as provided and were of analytical grade or better. Nuclear Magnetic Resonance. One- and two-dimensional NMR spectra were obtained on either a 300 MHz Varian Mercury spectrometer or a 400 MHz Bruker Advance 400 spectrometer, in D2O, referenced internally to the solvent. 195Pt NMR were externally referenced to K2PtCl4 (-1631 ppm; D2O, 25 oC,  = 21.4 MHz) at an operating frequency of 85.88 MHz. ADDIN EN.CITE  ADDIN EN.CITE.DATA 32, 33 1H NMR were run at 37 oC unless otherwise specified. For one-dimensional spectra, a spectral width of 7,000 Hz was used with 50,000 data points and a relaxation delay of 1 s. Two-dimensional nuclear Overhauser effect spectra (NOESY) were obtained using a 5,000 Hz spectral width with 256 increments in the t1 dimension, 2048 points in the t2 dimension and a mixing time of 0.35 s. Rotating frame Overhauser effect spectra were recorded with the same parameters as the NOESY, but with a mixing time of 0.5 s. Two-dimensional double quantum correlated spectroscopy experiments were recorded over a spectral width of 5,000 Hz using 256 increments in the t1 dimension, 2048 points in the t2 dimension and a pulse repetition delay of 3.0 s. All chemical shifts are reported in parts per million (ppm). Electrospray Ionisation Mass Spectrometry. Positive ion ESI mass spectra were acquired using either a Micromass Quattro Micro spectrometer or a Micromass Qtof2 spectrometer. Both spectrometers were equipped with Z-spray probes and were used to obtain spectra of solutions containing samples with concentrations ranging between 10 and 50 (M, that were injected into the instruments at a flow rate of 10 (L min-1. For the Quattro Micro spectrometer the source and desolvation temperatures were 150 and 120 C, respectively, while for the Qtof2 spectrometer these values were 60 and 150 C, respectively. The capillary tip potential and cone voltage were 2500 and 50 V, respectively, for the Quattro Micro spectrometer, and for the Qtof2 spectrometer these values were 2500 and 100 V. For both instruments between 10 and 50 acquisitions were summed to obtain spectra, which were calibrated against a standard CsI solution (750 mM) over the same m/z range. Theoretical isotopic distributions were determined using Mass Spec Calculator Professional v4.09. Growth Inhibition Assays. The murine leukaemia cancer cell line L1210 was grown in RPMI-1640 medium supplemented with 5% fetal bovine serum. Standard conditions for cell maintenance were used: 37 C humidified incubator with 5% CO2. Cells were plated in 96-well plates (100 L per well) at a concentration of 4 104 cells mL-1 in complete medium and then treated with a range of concentrations of drug in medium 100 L (0.00013 M to 50 M). The cells were incubated for 48 h under standard conditions before drug cytotoxicity was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) based growth inhibition assay. The assay was carried out by adding 50 L of MTT solution (5 mg mL-1) to the cell/drug mixtures and incubating for 2 h in standard conditions. The cells were harvested and mixed with 200 L of DMSO before the optical density (OD590) was read on a plate reader (MultisKan EX, LabSystems, Finland). Absorbance values at each drug concentration were plotted (y-axis) against the corresponding concentration of drug (x-axis) and the IC50 values were calculated from the resulting dose response curve. Data given are averages derived from between two to six independent experiments tested in duplicate or triplicate. Time course reactions. The metal complexes (1 mM) were reacted with either reduced L-glutathione or L-methionine (4 mM) in unbuffered D2O or phosphate buffered saline (NaCl 140 mM, Na2HPO4 8 mM, pH 7) in D2O at 37 (C and the 1H NMR spectra were obtained over a period of 7 days. All reactions were performed in triplicate. The time course experiments were also conducted under the conditions stated above but with the addition of CB[6], CB[7] or CB[8] (1 mM). Half-lifes (t1/2, h), defined as the time taken for 50% of the DNA intercalator to react with glutathione/methionine, were determined graphically by plotting elapsed time (h) versus percentage of unreacted species, measured from the change in relative integration of the non-degraded metal complex aromatic peaks. Synthesis of 5 ([bis-(-{reduced L-glutathione}bis{5,6-dimethyl-1,10-phenanthroline}bis{platinum(II)}]Cl2.H2O). [PtCl2(5,6-dimethyl-1,10-phenanthroline] (0.24 g, 0.5 mmol) ADDIN EN.CITE Fisher200717881788178817Fisher, Dianne M.Bednarski, Patrick J.Grunert, RenateTurner, PeterFenton, Ronald R.Aldrich-Wright, Janice R.<style face="normal" font="default" size="100%">Chiral platinum(II) metallointercalators with potent </style><style face="italic" font="default" size="100%">in vitro</style><style face="normal" font="default" size="100%"> cytotoxic activity</style>ChemMedChemChemMedChem488-4952 4200710.1002/cmdc.2006002116 in dimethylformamide (DMF, 25 mL) was added to a solution of AgNO3 (0.17 g, 1.0 mmol) in DMF (25 mL) and the resulting solution stirred overnight in the dark. The resultant precipitated AgCl was removed using a 0.45 m filter to yield a clear yellow filtrate. A solution of reduced L-glutathione (0.15 g, 0.5 mmol) in DMF (5 mL) was added, and the resulting solution stirred overnight in the dark. Diethyl ether (300 mL) was added to precipitate a pale yellow product which was collected by vacuum filtration and washed with diethyl ether. The yellow solid was stirred in water for 1 h. The insoluble impurities were removed by gravity filtration and the filtrate was loaded onto a Waters 2 g C18 reverse phase Sep-Pak, removing unreacted glutathione with water. The product was then eluted using acetonitrile:water (50:50) as a yellow solution and lyophilised. Anal. Cal c for C48H56Cl2N10O12Pt2S2.7H2O: C, 35.67; H, 4.37; N, 8.67%. Found: C, 35.26; H, 3.98; N, 8.28%. 1H NMR (300 MHz, D2O):  = 9.31 (d, J = 5.1 Hz, 2H, H2/H9), 8.96 (d, J = 8.5 Hz, 2H, H4/H7), 8.75 (d, J = 5.2 Hz, 2H, H2/H9), 8.66 (d, J = 8.5 Hz, 2H, H4/H7), 8.25 (dd, J = 5.2, 3.4 Hz, 2H, H3/H8), 7.75 (dd, J = 5.2, 3.4 Hz, 2H, H3/H8), 4.60 (t, J = 6.4 Hz, 2H, H52 ), 3.63 (m, 2H, H62 ), 3.44 (m, 2H, H62 ), 3.32 (m, 2H, H12 ), 2.72 (s, 6H, CH3), 2.64 (s, 6H, CH3), 2.16 (m, 4H, H42 ), 1.90 (m, 4H, H32 ), 1.66 (m, 4H, H22 ). 195Pt NMR (85 MHz, D2O):  = -2893 ppm (bs). ESI-MS: m/z calcd for C48H56N10O12Pt2S2+: 1418.28; found: 1418.0 [M-2H]2+, 709.1 [M-H]+. Reaction of 56MEGL with 52 -guanosine monophosphate. 52 -Guanosine monophosphate (2 mM) and 5 (1 mM) were incubated at 37 oC in 600 (L of unbuffered D2O and 1H NMR spectra were obtained at time periods of up to 7 days. Acknowledgements This work was supported by the University of Western Sydney Research Grant Scheme (No. 80582). Sharon Kemp was supported by a University of Western Sydney Postgraduate Award. We thank Assoc. Prof. Stephen Ralph (University of Wollongong) for help with mass spectrometry and Mr Shaoyu Wang (University of Western Sydney) for help with the growth inhibition assays. References  ADDIN EN.REFLIST 1. Brodie, C. R.; Collins, J. G.; Aldrich-Wright, J. R. DNA binding and biological activity of some platinum(II) intercalating compounds containing methyl-substituted 1,10-phenanthrolines. Dalton Trans. 2004, 1145-1152. 2. Jaramillo, D.; Buck, D. P.; Collins, J. G.; Fenton, R. R.; Stootman, F. H.; Wheate, N. J.; Aldrich-Wright, J. R. Synthesis, characterisation and biological activity of chiral platinum(II) complexes. Eur. J. Inorg. Chem. 2006, 839-849. 3. Wheate, N. J.; Brodie, C. R.; Collins, J. G.; Kemp, S.; Aldrich-Wright, J. R. DNA intercalators in cancer therapy: Inorganic and organic drugs and their spectroscopic tools of analysis. Mini Rev. Med. Chem. 2007, 7, 627-648. 4. 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A.; Patriarca, M.; Sadler, P. J. A novel dinuclear diaminoplatinum(II) glutathione macrochelate. Angew. Chem. Int. Ed. 1999, 38, 2949-2951. 14. Papadia, P.; Margiotta, N.; Bergamo, A.; Sava, G.; Natile, G. Platinum(II) complexes with antitumoral/antiviral aromatic heterocycles: Effect of glutathione upon in vitro cell growth inhibition. J. Med. Chem. 2005, 48, 3364-3371. 15. Mitchell, K. A.; Streveler, K. C.; Jensen, C. M. Isolation, reactivity, and molecular structure of bis(2,2'-bipyridine)(m-N-acetyl-L-cysteinato-S)diplatinum: model of the interaction of platinum with protein sulfur residues. Inorg. Chem. 1993, 32, 2608-2609. 16. Mitchell, K. A.; Jensen, C. M. Synthesis, characterization, and reactivity of platinum cysteinato and related thiolato complexes: Molecular structure of Pt2(.mu.-N-acetyl-L-cysteine-S)2(bpy)2. Inorg. Chem. 1995, 34, 4441-4446. 17. Kemp, S.; Wheate, N. J.; Wang, S.; Collins, J. G.; Ralph, S. F.; Day, A. I.; Higgins, V. J.; Aldrich-Wright, J. R. 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K.; Collins, J. G. Synthesis, cytotoxicity, cell uptake and DNA interstrand cross-linking of 4,4'-dipyrazolylmethane-linked multinuclear platinum anti-cancer complexes. Anti-Cancer Drug Des. 2001, 16, 91-98. 33. Wheate, N. J.; Collins, J. G. A 1H NMR study of the oligonucleotide binding of [(en)Pt((-dpzm)2Pt(en)]Cl4. J. Inorg. Biochem. 2000, 78, 313-320.  Table 1. Cytotoxicity (L1210 murine leukaemia cancer cell line) and reduced L-glutathione degradation half-lifes of four platinum(II)-based DNA intercalators. Cytotoxicity values represent IC50 in (M, which is defined as the required concentration of DNA intercalator to induce 50% inhibition of cell growth, and were determined from at least three experiments. Half-lifes (t1/2, h) represent the time taken for 50% of the DNA intercalators (1 mM) to react with reduced L-glutathione (4 mM) at 37 C and were determined from at least three experiments. Metal ComplexIC50 ((M)t1/2 (h)Cisplatin1.003.3a10.0092b6820.033c4030.463141.5020a. Data taken from reference  ADDIN EN.CITE Corden198722472247224717Corden, Brian J.Reaction of platinum(II) antitumor agents with sulfhydral compounds and the implications for nephrotoxicityInorg. Chim. ActaInorg. Chim. Acta125-130137198720. b. Data taken from reference  ADDIN EN.CITE Wheate200722382238223817Wheate, Nial J.Taleb, Robin I.Krause-Heuer, Anwen M.Cook, Rebekah L.h$CJmH sH h>2<h$5CJmH sH h$CJmH sH heh$CJH*mH sH  jhL$h$CJheh$6CJmH sH hb=8h$CJmH sH h$5CJmH sH heh$CJmH sH .*K,K/K=K>K?KAKDKKK:Lh$5CJmH sH heh$CJH*mH sH jh$CJUh$CJmH sH heh$CJH*mH sH heh$CJmH sH h$5CJmH sH hz>h$CJmH sH +6o DFxĊƊ$&46LNnʍ̍"23456UVY[ٽٽٽٽٽٽٯٽ٦ٯٌh$CJmH sH heh$56CJmH sH  jmh$CJheh$CJH*mH sH heh$CJH*mH sH heh$6CJmH sH heh$CJmH sH heh$5CJmH sH h$5CJmH sH 7[{|678Bďҏӏef֐056;<=󧚧xjjhIrGh$5CJmH sH hIrGh$CJmH sH h$5CJmH sH h$CJmH sH hShSCJmH sH hS5CJmH sH heh$5CJmH sH heh$CJH*mH sH  jmh$CJheh$CJH*mH sH heh$6CJmH sH heh$CJmH sH &8C&'`aLNSTKLXkldgd$=BCDFGtu AB pqst`acdvwyzQRTUqr./12DKߧZ[]^̬ͬ鹯鹯鹯鹯鹯鹯heh$CJH*mH sH h$CJmH sH jh$CJUhSh$CJmH sH h$5CJmH sH heh$6CJmH sH heh$CJmH sH hSCJmH sH ?ͬXY^_`٭ܭޭ XYZ[`abcɳʳX󙏙heh$CJH*mH sH h$CJmH sH jh$CJUheh$CJH*mH sH heh$6CJmH sH hIrGh$5CJmH sH hIrGh$CJmH sH heh$5CJmH sH h$5CJmH sH heh$CJmH sH 0XYZ[\ablm=Bbc~KLRUz{ھ۾ #`a¿ÿſƿȿʿ˿̿Ϳ޹ޫޡ޹޹ތޫ޹ތތ޹޹ޫޫޫޫޫޫh$5CJmH sH h$CJmH sH jh$CJUheh$CJH*mH sH heh$CJH*mH sH heh$6CJmH sH  jmh$CJheh$CJmH sH hS5CJmH sH hSCJmH sH 7Ϳοֿ׿ؿڿۿܿݿ޿߿>?fg=>.<= 䡴䡴t!j8Bh$CJUmH sH jh$CJUmH sH heh$6CJmH sH jh$CJU jmh$CJh$CJmH sH heh$5CJmH sH hIrGh$CJmH sH h$5CJmH sH heh$CJmH sH heh$CJH*mH sH -  12 '('(<=>?HIJKLX~޴ޣ޴ޒwheh$5CJmH sH heh$CJmH sH !j`h$CJUmH sH !jNQh$CJUmH sH jh$CJUmH sH heh$6CJmH sH heh$CJH*mH sH h$CJmH sH heh$CJmH sH jh$CJU.~_`ef[\^l䴪䠖͠䠖͠heh$CJH*mH sH h$CJmH sH jh$CJUhSCJmH sH heh$5CJmH sH h$5CJmH sH heh$CJH*mH sH  jmh$CJheh$CJmH sH heh$6CJmH sH 5GJpquvjlRTf佲ֽxxohL$h$CJ jmh$CJheh$5CJmH sH $j kh$CJH*UmH sH jh$CJH*UmH sH h$CJH*mH sH jh[&h$CJH*Uh[&h$6CJheh$CJH*mH sH heh$CJmH sH heh$CJH*mH sH +?@K;<*+-."#mngd$dgd$fj 68HJ:<ptbd%'./?@bcG俚}heh$56CJmH sH  jmhUSh$5CJhf5CJmH sH  jhL$h$CJheh$6CJmH sH heh$5CJmH sH heh$CJH*mH sH hL$h$CJheh$CJmH sH heh$CJH*mH sH -GIJLMNQVW23                    z |         F H  ƸƮƸƈƖƸƸƸƸƸƸƸƸƖƸ~ƈƈƈhch$6CJheh$6CJmH sH heh$CJH*mH sH h$CJmH sH jh$CJUheh$CJH*mH sH heh$CJmH sH heh$5CJmH sH hfhf5CJH*mH sH hfhf5CJmH sH 1      f h "$HJ,2JNPTVZ\`dfhjl8?K jmh$CJh$5CJmH sH heh$5CJmH sH hch$6CJheh$CJH*mH sH heh$CJH*mH sH heh$CJmH sH heh$6CJmH sH 8KL^_*.;<*+-."#LW\mn*PUbcMX]mn #(ʼزʼزʼزʼزʼزʼززʼزʼزʼزʼزʼزʼزʼh$CJmH sH hx[h$5CJmH sH hx[h$6CJmH sH hx[h$CJmH sH heh$CJmH sH jh$mh$CJUFbcmn89#$+ , !!""""##$$%%%%gd$(89#$   + ,           !!!!!!"""""""#####n$$$$$$$$%%n%z%%%%&&'''e'p'u'''((Y(g(k(x(y((((hx[h$CJH*mH sH hx[h$5CJmH sH hx[h$6CJmH sH h$CJmH sH hx[h$CJmH sH K%''''x(y( ) )))**++a,b,--.....00dgd$ 0^`0gd$gd$(() ) )x)y)z){)|)}))))))))))))))))*****h+x+}+++?,L,Q,a,b,,,,-----..*.+.a.b.h.i.r.s.u....־־ֱ־־־־ֱ֣֣־־hx[h$CJH*mH sH  jmh}|CJmH sH hx[h$CJH*mH sH h$CJmH sH hx[h$CJmH sH hx[h$5CJmH sH hx[h$6CJmH sH >.......^/`/d/e/00u0v000000000000000ۿ۱ۥ۱ۿ۞{ocZTM h$CJH* h$CJhL$h$CJhO^h$5CJH*hHh$5CJH* jmh53h$5CJhbe,h$5CJH*h53h$5CJ h$5CJ jmhL$h$CJheh$CJH*mH sH heh$6CJmH sH heh$5CJmH sH heh$CJmH sH jh$mh$CJUheh$mH sH 00000000ikdt$$IfFF & t6    44 layt$ d$Ifgd$0011111 1 1111111111&1'1(111N1O1#4$4&4'4F4G45:;LMNOSTr췪{wh-Ljh-LUUjhash$CJUheh$CJH*mH sH h$CJmH sH jhM;h$CJUheh$CJmH sH hc&`h$CJH* h$CJH* h$CJh\h$5CJ h$5CJhL$h$CJhHh$CJH*,011 1 1 d$Ifgd$ikd#u$$IfFF & t6    44 layt$ 11111 d$Ifgd$ikdu$$IfFF & t6    44 layt$111"1%1 d$Ifgd$ikd'v$$IfFF & t6    44 layt$%1&1(1-101 d$Ifgd$ikdv$$IfFF & t6    44 layt$0111QSvLMP $da$gd$dgd$ikdw$$IfFF & t6    44 layt$r>Wang, ShaoyuHiggins, Vincent J.Aldrich-Wright, Janice R.Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehiclesDalton Trans.Dalton Trans.5055-5064200710.1039/b704973k5. c. Data taken from reference  ADDIN EN.CITE Kemp200721692169216917Kemp, SharonWheate, Nial J.Buck, Damian P.Nikac, MaricaCollins, J. GrantAldrich-Wright, Janice R.The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalatorsJ. Inorg. Biochem.J. Inorg. Biochem.1049-105810172007DOI: 10.1016/j.inorgbio.2007.04.0094.  EMBED ChemDraw.Document.6.0  Figure 1. The chemical structures of (a) 1 (S,S-dach and R=CH3), 2 (R,R-dach and R=CH3), 3 (S,S-dach and R=H); (b) 4; (c) reduced L-glutathione; and (d) L-methionine, showing the proton numbering scheme used. * Indicates a chiral centre, either R or S. Anions have been omitted for clarity but are generally chlorides.  Figure 2. The 1H NMR spectra of the reaction of 4 (1 mM) with reduced L-glutathione (4 mM) at 37 oC showing the production of the major degradation product 5 (%) and other minor product(s) (%). These spectra are indicative of those obtained for 1 and 3. The spectra of 2 are considerably more complicated due to the lack of phenathroline symmetry because of the presence of only a single methyl group.  Figure 3. The 195Pt NMR spectrum of the reaction mixture of 2 72 h after reaction with four mole equivalents of reduced L-glutathione, showing unreacted 2 (-2828 ppm), its doubly bridged dinuclear platinum(II) degradation product 5 (-2916 ppm) and a proposed mononuclear degradation product at -3551 ppm.  EMBED ChemDraw.Document.6.0  Figure 4. The proposed chemical structures of the three degradation products observed from the reaction of 1 with reduced L-glutathione from the analysis of the electrospray ionisation mass spectrum. Charges, counter ions and the chirality of bonds have been omitted for clarity.  Figure 5. The (a) 1H and (b) 195Pt NMR spectra of 5, the major reduced L-glutathione degradation product of this family of platinum(II)-based DNA intercalators. The 1H NMR spectrum of 5 is identical to the major degradation product indicated in Figure 2.  EMBED ChemDraw.Document.6.0  Figure 6. The proposed chemical structure of 5 showing the two possible isomers (syn and anti), with respect to the glycine residue. These isomers disrupt the symmetry of the dinuclear complex thereby giving two sets of phenanthroline and glutathione resonances in the 1H NMR spectrum. This figure is a modified version of the one given in reference  ADDIN EN.CITE Papadia200522402240224017Papadia, ParideMargiotta, NicolaBergamo, AlbertaSava, GianniNatile, GiovanniPlatinum(II) complexes with antitumoral/antiviral aromatic heterocycles: Effect of glutathione upon in vitro cell growth inhibitionJ. Med. Chem.J. Med. Chem.3364-337148200514.  EMBED ChemDraw.Document.6.0   Figure 7. The chemical structure of cucurbit[n]uril (where n = 5, 6, 7, 8, or 10), and a model of CB[7] demonstrating the barrel shape and internal cavity of cucurbit[nrstuvˎ̎ΎώЎюӎԎՎ֎ùzpf[p[pph$5CJmH sH h$CJmH sH h`,CJmH sH h`,h`,CJmH sH h}|h}|CJH*mH sH h}|h}|6CJmH sH h`,5CJmH sH h}|5CJmH sH h}|CJmH sH heh$CJmH sH heh$5CJmH sH  h$CJjh-LUjwh-LUjdK h-LUV#lmqrďȏɏ68^`jlM䴮zi!heh$B*CJmH phsH h`,CJmH sH !heh$B*CJmH phsH h`,5CJmH sH heh$CJH*mH sH  h$CJjh#h$CJUh}|CJmH sH heh$6CJmH sH h$5CJmH sH heh$CJmH sH heh$5CJmH sH )MNOPZ]aȒɒ5678!"”Ô͔Ք֔εΧεΟ|vεΧivjhv h$CJU h$CJjh$U-j1cK h$PJUVaJmH nH sH tH h$jh$Uheh$6CJmH sH h$5CJmH sH heh$CJH*mH sH heh$CJmH sH  h$5CJjhMVh$5CJUheh$5CJmH sH (Ô•Õƨh]hgd$ &`#$gd$gd$ $da$gd$dgd$  hi{|•ĕŕ8;@DEF窦sf\ffh$CJmH sH jhM;h$CJUheh$5CJmH sH  h$CJj h$U-jvJ h$PJUVaJmH nH sH tH h$jh$Uh`,h$5CJmH sH h$5CJmH sH heh$CJH*mH sH heh$6CJmH sH heh$CJmH sH h`,5CJmH sH !ӚԚ՚֚ߚƨǨŷũũũŠڜ{sosososoe_e_ h`,0Jjh`,0JUh jh Uhvh!h$5CJjQh$UjJ h$UVh$ h$5CJUheh$6CJmH sH heh$5CJmH sH heh$CJmH sH j3(h$Ujh$Ujh$Uj,I h$UVheh$mH sH %]urils within which platinum(II)-based drugs can be partially encapsulated and slowly released, preventing their degradation by thiol amino acids, peptides and proteins. Table of Contents Graphic  EMBED ChemDraw.Document.6.0      PAGE  PAGE 26  $h]ha$gd$ &`#$gd$    hvh h`,0JmHnHu h`,0Jjh`,0JUh`, A0P&P 1h/R :p$. A!"S#$% DBrodie200419511951195117Craig R. BrodieJ. Grant CollinsJanice R. Aldrich-WrightDNA binding and biological activity of some platinum(II) intercalating compounds containing methyl-substituted 1,10-phenanthrolinesDalton Trans.Dalton Trans.1145-11522004Jaramillo200611031103110317Jaramillo, DavidBuck, Damian P.Collins, J. GrantFenton, Ronald R.Stootman, Frank H.Wheate, Nial J.Aldrich-Wright, Janice R.Synthesis, characterisation and biological activity of chiral platinum(II) complexesEur. J. Inorg. Chem.839-8492006Wheate200718531853185317Wheate, Nial J.Brodie, Craig R.Collins, J. GrantKemp, SharonAldrich-Wright, Janice R.DNA intercalators in cancer therapy: Inorganic and organic drugs and their spectroscopic tools of analysisMini Rev. Med. Chem.Mini Rev. Med. Chem.627-648762007Kemp200721692169216917Kemp, SharonWheate, Nial J.Buck, Damian P.Nikac, MaricaCollins, J. GrantAldrich-Wright, Janice R.The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalatorsJ. Inorg. Biochem.J. Inorg. Biochem.1049-105810172007DOI: 10.1016/j.inorgbio.2007.04.009Wheate200722382238223817Wheate, Nial J.Taleb, Robin I.Krause-Heuer, Anwen M.Cook, Rebekah L.Wang, ShaoyuHiggins, Vincent J.Aldrich-Wright, Janice R.Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehiclesDalton Trans.Dalton Trans.5055-5064200710.1039/b704973kFisher200717881788178817Fisher, Dianne M.Bednarski, Patrick J.Grunert, RenateTurner, PeterFenton, Ronald R.Aldrich-Wright, Janice R.<style face="normal" font="default" size="100%">Chiral platinum(II) metallointercalators with potent </style><style face="italic" font="default" size="100%">in vitro</style><style face="normal" font="default" size="100%"> cytotoxic activity</style>ChemMedChemChemMedChem488-4952 4200710.1002/cmdc.200600211DKemp200721692169216917Kemp, SharonWheate, Nial J.Buck, Damian P.Nikac, MaricaCollins, J. 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Aldrich-WrightDNA binding and biological activity of some platinum(II) intercalating compounds containing methyl-substituted 1,10-phenanthrolinesDalton Trans.Dalton Trans.1145-11522004Jaramillo200611031103110317Jaramillo, DavidBuck, Damian P.Collins, J. GrantFenton, Ronald R.Stootman, Frank H.Wheate, Nial J.Aldrich-Wright, Janice R.Synthesis, characterisation and biological activity of chiral platinum(II) complexesEur. J. Inorg. Chem.839-8492006Wheate200718531853185317Wheate, Nial J.Brodie, Craig R.Collins, J. GrantKemp, SharonAldrich-Wright, Janice R.DNA intercalators in cancer therapy: Inorganic and organic drugs and their spectroscopic tools of analysisMini Rev. Med. Chem.Mini Rev. Med. 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J.Kim, S. Y.Ko, Y. H.Sakamoto, S.Yamaguchi, K.Kim, K.Novel molecular drug carrier: encapsulation of oxaliplatin in cucurbit[7]uril and its effects on stability and reactivity of the drugOrg. Biomol. Chem.Org. Biomol. Chem.2122-21253112005ISI:000229373500014<Go to ISI>://000229373500014 10.1039/b504487a DWheate200722382238223817Wheate, Nial J.Taleb, Robin I.Krause-Heuer, Anwen M.Cook, Rebekah L.Wang, ShaoyuHiggins, Vincent J.Aldrich-Wright, Janice R.Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehiclesDalton Trans.Dalton Trans.5055-5064200710.1039/b704973kTaleb200721652165216517Taleb, Robin I.Jaramillo, DavidWheate, Nial J.Aldrich-Wright, Janice R.Synthesis of DNA sequence selective hairpin polyamide platinum complexesChem. - Eur. J.Chem. - Eur. J.3177-3186132007Jaramillo200616081608160817Jaramillo, DavidWheate, Nial J.Ralph, Stephen F.Howard, Warren A.Tor, YitzhakAldrich-Wright, Janice R.Polyamide platinum anticancer complexes designed to target specific DNA sequencesInorg. Chem.Inorg. Chem.6004-60134515200610.1021/ic060383nz DWheate200166617Wheate, N. J.Cullinane, C.Webster, L. K.Collins, J. G.Synthesis, cytotoxicity, cell uptake and DNA interstrand cross-linking of 4,4'-dipyrazolylmethane-linked multinuclear platinum anti-cancer complexesAnti-Cancer Drug Des.Anti-Cancer Drug Des.91-981622001AprISI:000174796900003<Go to ISI>://000174796900003 Wheate200011111117Wheate, N. J.Collins, J. G.<style face="normal" font="default" size="100%">A </style><style face="superscript" font="default" size="100%">1</style><style face="normal" font="default" size="100%">H NMR study of the oligonucleotide binding of [(en)Pt(</style><style face="normal" font="Symbol" charset="2" size="100%">m</style><style face="normal" font="default" size="100%">-dpzm)</style><style face="subscript" font="default" size="100%">2</style><style face="normal" font="default" size="100%">Pt(en)]Cl</style><style face="subscript" font="default" size="100%">4</style>J. Inorg. Biochem.J. Inorg. 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(c)Qr'L &ChemDraw can't interpret this label. 2R{ sڶ}ˌ &ChemDraw can't interpret this label. 3S[gˌ &ChemDraw can't interpret this label. 4Tr?8?E &ChemDraw can't interpret this label. 7Ur"'$ˌ &ChemDraw can't interpret this label. 8V??̌ &ChemDraw can't interpret this label. 9WwLoLyeT &ChemDraw can't interpret this label.1'X2?uۘ?ue&} &ChemDraw can't interpret this label.2'Y2 ze &ChemDraw can't interpret this label.3'Z2??e& &ChemDraw can't interpret this label.4'[[gef &ChemDraw can't interpret this label.5'\2 e &ChemDraw can't interpret this label.6']xבY3Y ] &ChemDraw can't interpret this label. *^x3S` &ChemDraw can't interpret this label. *_x3Ӛ &ChemDraw can't interpret this label. *Ld)??" FEMF+@ ObjInfo CONTENTS+=_1264841009 !mA. pspsOle VjCD0100ChemDraw 11.0 sUU  @@' :;E<  BCD a  3xx# $$$$D $ArialTimes New RomanxXXg{XXdd'`c33 $$s|2}7zt|2}$7z |2}$z M 7 }҂ 7 $΍.hh 7 Zi  /An atom in this label has an invalid valence.+&xexe&l # aN .D      e  s  *i  yo  ROj /An atom in this label has an invalid valence.+RfzZRfm # aN K /j 7 m 7 #e'I 7  T $ N+ Ta<OaoY #a Pt ۪(? '/An atom in this label has an invalid valence.+7J֐; $֐;dȮF #aaNH2 &j@ (/An atom in this label has an invalid valence.+7j<,j<p6G #aaNH2 4ժ{ +7 xָ -7 c% /7 tc% 07  \.      \.   \.     \.   \.     \.   \.   \.     \.   \.     \.   \.    \.     !\.   "\.     #\.   %\.   &\.   )\.   *\.   ,\.   .\.   1\.   2\.   3\.   U5fh}/+fh}7 &ChemDraw can't interpret this label. (b)u3}-\N 3}-\O q 7P }> 7Q oqi 7R Z| /An atom in this label has an invalid valence.+&& # aNS .i T  U ek V sN W * <96   number.Y{H 6X y, <95   V8 snumber wx ӕ 5Y v /An atom in this label has an invalid valence.+v#zZv#V # aNZ K ћ 7[ m~ 7\ #e7 7] xx  N+xxaasar} #a Pt^ ۪c /An atom in this label has an invalid valence.+7J_ $_dȮYk #aaNH2_ &d /An atom in this label has an invalid valence.+7`,`p[Nl #aaNH2` 4ժD 7a x$ +73RD((D-\ # aRb tܟ 7c ܟ 7d b. 7e I #   k/iqueryru9Bxu9BrF *f tI #   Jq \oqueryBP?BpP?BBF *g pЫ. 7h \.O P  m i i \.P Q j \.Q R  i x k k \.R S l \.S T  k n r m m \.O T n \.S U o \.U V  n s w p p \.V W q \.W X  p } | r r \.T X s \.U Y t \.Y Z  s y u u \.Z [ v \.[ \  u w w \.V \ x \.R ] y \.] Y z \.] ^ { \._ ] | \.X ` } \.W a ~ \.b c  \.c d  \.d e  \.e f  \.f g  \.g b  \.^ f  \._ e [xQћU|Qћ`|Q :7|`H ;7ћ`H =7 <\.  >\. ([x(ނU ?&ChemDraw can't interpret this label. 2+[QwћS|ћ | 47|H  57ћH  77 6\.  8\. ӨR[QwR(^S 9&ChemDraw can't interpret this label. 2+ p[!rpg@ @kt[ A01k[% C+7X[![!]r[f) # aOYg@ E+7ZFg<Zg<_K #aaOHv G7s!I I+7pw!E p!EpwTL # aHL- V' k70%B l +7 >ʠ>L$:E # a S3 Q2] n76>x p79 KK r01;>GJ<99L t?A v+7sY3YL #aaOHB=89L x+7 %9d 9d>9$ # aOEwu z+7d E>}z  #aaNH2H"4 |+74r4g # aH2 m\.-0 5 o\.03 8 q\.36 ; s\.69 > u\.9< A w\.<? D y\.<BG {\.9E J }\.9H N3fh(fh52 ~&ChemDraw can't interpret this label. (a)OffU &ChemDraw can't interpret this label. (d)PffU25 &ChemDraw can't interpret this label. 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$!ڒTm.BygQ3_Z R*Վ{`)|:[RJD*PŲZ*Gb/ -T"-GbֶX;[jXU,tl1ZXUՖfz8NsmY2#J1W׬T/@b- _Fb޳$J1W׬T/Q҄kBR{D)Yg[M*c}`)tQ( RږiӽJmZ #J1חfhK3XU>[[6ëԦ@>s}Yh)wNc}T)lm=LR)ReYZ<-:9Q복jJmf #J1׷Pc}T)LJmV #J1חdf{ZFQ3[L( R%n6;y뫄DKfBY0Gb/)񺴺Zs" 1ז$z]ZgQyz^R%y^6oM.Gb*oѻ}Y0Gb/)ڼ8RPLTR~˞c}T)oVWKRu-Z!]M$z>s}mb Q`)"[[(-L.Gb֯-z\la|,\_YiR^B>s}Ifׯ-[vQ복 jKkw}4)Ro@[&J1gkE)5jXV2mjЋQ3$2"R#.VWQ3NVb}T)lmIkTF]oJ1ח4[2oTQK:]rqH^*\_JDb#>s}IcP`J/G}*գ IJZ|͓+\ K}/ט;\)Rz:zJ_c}T)%\-7r/\-\mP*8Re~m@ RURRRe͔z}Xs}Ig@V6 r}^*\_* ZM,8p3>WR}^WzԐ[j5Os}YhGzTjA/Ua&_ J1݆4T*YnMzچRԳ'<*J1חdFnQ-~Wѧx=5 \)̨׫RQF,`)̘ׯJ#R!QJ ڨJ1חtƽAmL*XU)eM$Z-s}$2&JcQb/F~nz>󼉔^|Gb-IFVK1:su)eM$Z-żgIbu3F5XU$WURΰ)b}T):oX[[[_K1g:ZoX[ӋQ[1TNXUc ʀKb}T)L$m&VRe:E_NlЋqg߀JIJZW>zD)[1qg_JIJZV$s}Y蔎nQV"qg_JuIJZV$u}=SJH`)ٗtNlxq복 hyکq,\_+_ RچaJ 5_uK1חު iްJ1gkD*5bY.`)۴Q+oLQ복KE*5bY3`)۵qz\Q?U۬d^smYtm!u}Ig@/(*XUz kju-+7҄kKVK1: j\,Gb/ɼ&J1W-nHl#R%w{ڻb}T)li҈(K1חt kVJ/Gb֖DFEitwwR%z#{b}T)8'RzsmI}ވj)YGbn"ϣ 1ז$hb޳$J1:[[  XoT{ߺ^*n"0iB̵%3QR̳(\DJY/> 1ז$>jb޳$J1:F3jz>s}ICވ*`)uuzIb- }XiX\gQfBܵ%FR{D)YtcH7ҠJ1a.>5 k>s}I#Ҁ(5Vz>Tj5?ĕb/ }_U# zw\)7Rgj5+E]@!J 6}4~ XzǴA7RF1JXV;3yBFULoLQ복%qwX, V1o\QK:}\5֠J1q+>M$J1٬d=WuIS(B{Ѥg]TLjIoA/Gb*5Z|ZH,ɕb/|cX7ҠJQϾ6Xv[󼬔hs}IoD;oM/Gbt^*\_8Ω_uKQ7\ޒbW]R%z#ڹ^*\mLXعqK:ycgUҋQ복-VRjygQ`)rGbτVixu8RԳoh..ӚVk>s}I粎ڥުXUzZ -Gb/\1]-oЋQg߸hךƵXUaroA/G}#1R|Akb}T):~QJ kb}T)7R_kbYm ևbτ^QJkz>S/i5J1Y2y>3/k)YǓbY2hB̵e+^%Qye+:zDGܠJ1eU-zjbY7ևb/ ]-J*uE^o+E=zJ)ӪIJZO+E]_kRRg__q׫]\*E]_ԮjbyW$o@JR3XU>[ې4,J!WJaK:_\J1gkE)5 wR%1*WRVH+E)ArR%y+UҋQ복VRjU`)5*k*WRJe_U|J1ח$VjĮU /ɬѮ*k'X$s]SזrY/GbvvW]),\_[]']\*\zDkw}4) uCompObj ~ObjInfoCONTENTSzP_1241937650!mA. pspsVjCD0100ChemDraw 11.0,Microsoft Word - Glutathione 18 Feb 08.docCdкE g M :;E<  BCD a  3xx# $$$$2$ArialTimes New RomanxXXg{XXd'`&H33 $$Cd3-ECd3-E Cd3-ECd3-E 9K G F+7g&O CF C@O J # aO g_ G G hT5 H+7lTE2#dTE21ux9 #aNH g_$ I7 h J | K+7g1x #aOH g_3l L+73b3}Z3 c3D # aO h(X M |(X N7 wNi O+7͌wfxwf4ݬl # aS g_wNi P+73bweZwe3bt #aNH 9KwNi Q 3SA(X R7 g-(X S7 #vNi T g-,tz U+73s1,v),v3,l #aNH2 4vNi V ,tz W+7 ,vDd,v3 _ #aOH (X X+7 @TCd@T2 \ # aO 4SA,tz Y+7@E,v<,vYE,L~ # aO t{p* m7 g!X n7 N:q= o7 τ  p/An atom in this label has an invalid valence.+NrdrN! # aN A6* q x r uˡ y : z A {  | >Ԏ /An atom in this label has an invalid valence.+ ޯ<< ޯo # aN s 7 f 7 оV' 7 ,A N+ANHz; #aPt LEA 7 L>- 76T j 0/An atom in this label has an invalid valence.+7#Tm̩T[ITm #aNH2=b 2*t 487}L 67)qH 77imZ 87/Vp 97 1V, ?+7sVVdV$#aNH2 Z   [   \   ]   ^   _   `   a   b   c   d   e   f   g   h   i   j   k   l   s     t   u       v   w        x   }   ~                                                    /   1   3  5  :  ;  <  =  >   @  X/X /X/X h /An atom in this label has an invalid valence.+ݎeFt}ݎeC5l # aS }DZh /An atom in this label has an invalid valence.+I1^ݎeUݎeW^C5l # aS hiEݫ 7 ];* 7 (FN< 7 ;YR /An atom in this label has an invalid valence.+]RbUR]B # aN E;cFԘ   Y  v/  fVs  (vJ  )bߺ  S q /An atom in this label has an invalid valence.+ل|ل  # aN t] 7 t(8 7 ު 7  nx N+[qNuiNurB #aPt X 7 } 7hiE$ 7];iQ6 7(FEG 7;YBF /An atom in this label has an invalid valence.+]BCbUBC]uFJ # aNE;cM~5  Y$ vd5 fV#  (v !)bs "S E /An atom in this label has an invalid valence.+ IB| IB<|I # aN#E 7$t(\A4 7%s# 7& nU N+[qUi&PrYyZ #aPt'X@ 7(T 7I*Cm\ O!Fm +7FjEFjz$y;q #aNHQ*J~ S!L 7U*O9 7W!Rl Y*V [!X +7z$X%X%X # aO]!@:K _*=: +7.=o6&=o6:7p= #aNHa!;( 7c*7 e!4n +7z$4%4%4F  # aO+ @:K +7R@RG@RG1@O # aO>7 +7B7cI:7RBj" #aOH>J~ +7BJzcI:JzRBJ # aO+ Rl +7RRԮRԮ>Rd #aNH2>V +7BVcI:VRB #aOH(MA"I\ 7RDm\ #@Gm  +7,GjxնGjgz;q #aNHRJ~  #@L  7RO9 7#@Sl RV #@X +7,XXFX # aO#@@:K R=: +7c=o6돤=o6>p= #aNH#@;( 7R8 #@4n +7,44F4F  # aOL@:K !+7 @RGÓ@RG"@O # aOu8 #+7b8\8K|k" #aOHuJ~ %+7bJz\JzK|J # aOLSl '+7 SԮSԮ/Sd #aNH2uV )+7bV\VK| #aOH"I\ +7                                                                                        )  .**  +  *,9,  -  /,.3.  /  0  4/181   2  ! <13;3 ! 4 " 5 "# :466 #$ 7 $% 688 % 9 & : &" ; 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\C3֮hߊEھIi&}Y0nQUEe)-trmE[EWD(z)^-:][]h*mFVeoO7ΫE'[#(ڻ"B"R7^޲!E[EWD(z^ëE~w[]hRro/>U&uc[j-E[EWD(Z 5olЪhEYijm_h)x#ݜj?nYoEI@iD5PЉ^֍+"}` j-VE{WD(Z&yfQUEI6 ޭӲ{SEE{W(䦹Do?(ڻ"B$w[46ZFVgy՚w*ڻ"BѤvݶRͲQUE,ٝ`HWvjnE,zӼehhE$A^m_hCow{tlm_FOyӌ߇ns+bMr;T-qEjwD+"-z;ehhE9(`ޠ\(*ڿ"B$wpownO6qǓxy'ޠ8/owEEU!_1 }[ݹ-wnjO fuU.kҢXWDx|#ɱ8Y.Qt+"ʓEv'Ӧ"m5'n]E!IV/P<;D7wU(ڿ"BYnD+"-z3CtQUEfm_hLlm_FO՚(,ڻ"ϛVV[|jP7SCxG=z^yO=FمSCu/]fkXjy՚[]hRn^ijm_h)x/޽uU^-P4G'ijm_hې6 #zY7P4 W7kQ]wEEowe(*ڿ"BMӍjщ7,Z]hW7^޷!E[EWD(~ziCIL&Ev1:I6:E(ڻ"B$w(f+"-Ϥ&AL?ōnUwEIY+"-o=@Wk?E+"ېϓhhE,2Mx]EEA5eQUEgjƫEWD$`ކxōn}+"-o=XW>(^u(ڿ"BY!M$'Q]wEEo.m6 T+"`+ʛmhfѹ(,ڿ"BY/ ʚsQYE-֯)]hћ=T/E[EWĸ"p5hF{WD(ޛ=L7'E[EWĸSIj|oPsE7ڻ"B$>(*ڿ"ƍdGvF{WD(蓍+b$5V>79Ƌ]h{ToH]O6q;,xho$+#GfE7ڻ"F#Uѝ$IѦjm_Fޣy(]EE{WD(Z =Z7'(ڿ"BzCuEE{WD(Zn6Z.EWD(ۇX~L{ThhE?J3ާҽYm_h|nO^S175Z]hћ=F'zY7Pc{a)^-:]Q]wEEnXT؆lm_ztZt<,ڪhEOTWe: Ҧ4ӪhEuSUxlm_h)xx]ݫVE{WD(Ԟ=>q> c ? d >IH@@ e A j B k AGCC l D m CEE n F p JG q H s I u J v  FK w  L ~  QMM  ! N !" MO\O "# P #$ ROQVQ $ R #% S %& WRT[T &' U '( _TV^V $( W %) X )* ]WYY *+ Z +, Y[[ &, \ "- ] -) ^ (. _ '/ ` -  a -  b  0 c 1  d 02 e 03 f 14 g 15 h3f/ti3i  7j 3i  74wR2 ٣{c o| p01 q+w{# aC| r+zwz5#aNH s+ w Щ@# aOyI tf / uf / v+f+wf+Щ 2# aOR w+R2wR2 R2#aOH c x  y z { | } ~   @3f3fY/t/An atom in this label has an invalid valence.#aGlu  ij "K0IQg; \03[  78/8)T +78O;IQ85IQg;lW# aH  EyuI_e|3s} +73zEy3z4_# aH8u 7  " 1uk=f883s} 75ue̟̟3 9ij +99IӢKW# aC9q +tht#aNH,d + ͼ ͼ## aOui      8<3cz853czk=f/An atom in this label has an invalid valence.#aGlye1u 7  ylfJɄ\ 8o3s\ 7!|T 76 Vv Y! pm ZP6 4g +44Y!#aNH/s%e3h 77;4"3km9;4H ;4 5 01;4"n +;T"3k;"3kmp# aC;45 +;T5;Tl5#aNH;46"n +;T9l;3lm9"r# aO;4 [ ;4n ;46n +;T9n;3nm9# aO;4 +;T ;m #aOH;4H           3eE3e>/s/An atom in this label has an invalid valence.#aGlu'3h 7( %' )3cfes*e3si 7+3si 78R #{flb  m~| !01zxyny "+7P{f# aCo~| #+7yyO58#aNHpy $+7⌑⌑P# aOqyH %rf. &sf. '+7fm+fm+P1# aOtR (+7RR PR#aOHu b ) v *uow +npx ,moy -mnz .lm{ /lq| 0qr} 1rs~ 2rt3cf3cfs/An atom in this label has an invalid valence.#aGlu *+ "l&Ik$[%33[ 78S 79xv\Y!\pm 6 9g +Y0rY!#aNHr3sh 7;:"j Z:H <:5 =01:"m >+:"j:"jp# aC:5 ?+:5n:~5n#aNH:"m @+: ?l:?l "sr# aO:[ A:n B:n C+: n_:n_ # aO:z D+:Z:ZZ#aOH:H E  F G H I J K L M N3ceŔ3ce>r/An atom in this label has an invalid valence.#aGlu 3sh 7  3B> VB3 &ChemDraw can't interpret this label.&syn conformation3B>?3BfƝ &ChemDraw can't interpret this label.'anti conformation0e(3sz#33i   13sze33i   3ye 3h   e}3yx3h                 ;            ! 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LPN8 %  % ( % ( &% / 6  % &% (  ?  % ( '%  % ( % &%  6  % &% (  ?  % ( '%  % ( % &% { 6  % &% (  ?  % ( '%  % ( % &%  6{  % &% (  ?  % ( '%  % ( % &%  6  % &% (  ?  % ( '%  % ( % &% )6  % &% (  ?  % ( '% % %  '% ( &% %  TTBQm†@1@LPO? %  % ( % ( &% 6) % &% (   % ( '% &% 6  % ( '% (  % ( % &% > 6  % &% (   % ( '% % %  %  '% ( &%  %   TT9PA_m†@1@ LPN8 % %  % ( % (  =%  = TT9[Ajm†@1@ =LPH8 % &%  6>  % &% (   = % ( '%  % ( % &%  6  % &% (   = % ( '%  % ( % &% 6  % &% (   = % ( '% % %  '% ( &% %  TTBQm†@1@LPO? %  % ( % ( &% /6 % &% (   % ( '% &% %6  % ( '% ( %  %  '% ( &%  %   TTP(Y7m†@1@ LPO? %  % ( % ( &% 4 64  % &% (    % ( '% &% H 6H    % ( '% ( % % p%  '% ( &% p% p TT jym†@1@pLPO? % %  % ( % ( %  TTjym†@1@LPH8 % &% 6P % &% (   % ( '% %  %  '% ( &%  %   TTjym†@1@ LPO? %  % ( % ( &%  6 P % &% (    % ( '% &%  6 P   % ( '% ( % % | %  '% ( &% | % |  TT(7m†@1@| LPN8 % %  % ( % (  %   TT(7m†@1@ LPH8 % %  %   TT.9m†@1@ LP2& % % &%  6  % &% (    % ( '% % %  %  '% ( &%  %   TTjym†@1@ LPO? % %  % ( % ( J% J TTjym†@1@JLPH8 % &% )6)P % &% (  J % ( '%  % ( % &%  86  % &% (  J % ( '%  % ( % &%  6  % &% (  J % ( '% % % ? %  '% ( &% ? % ?  TT~m†@1@? LPN8 % %  % ( % ( ? % ?  TT~*m†@1@? LPH8 % &% 1 p 6 5  % &% (  ?  % ( '%  % ( % &%  6 6 p  % &% (  ?  % ( '%  % ( % &% { 6 6  % &% (  ?  % ( '%  % ( % &%  6 6{  % &% (  ?  % ( '%  % ( % &%  6 6  % &% (  ?  % ( '%  % ( % &% )6 6  % &% (  ?  % ( '% %  %  '% ( &%  %   TTm†@1@ LPO? %  % ( % ( &% { 6<  % &% (    % ( '% &% i 6)*    % ( '% (  % ( % &% > 6 6  % &% (    % ( '% % %   %  % ( %   %    TT9Am†@1@  LPH8 % %  '% &%  W %  W  TT9Am†@1@ W LPN8 %  % ( % ( &%  Q 6>  % &% (   W  % ( '%  % ( % &%  6 Q  % &% (   W  % ( '%  % ( % &% 6 6  % &% (   W  % ( '% %  %  '% ( &%  %   TTm†@1@ LPO? %  % ( % ( &% "j 6*  % &% (    % ( '% &% -| 6<    % ( '% ( %  O %  '% ( &%  O %  O  TTP*Y9m†@1@ O LPO? %  % ( % ( &% H 6H   % &% (   O  % ( '% &% 4 64    O  % ( '% ( % % p%  '% ( &% p% p TT m†@1@pLPO? % %  % ( % ( %  TTm†@1@LPH8 % &% 6 6 % &% (   % ( '% %  %  '% ( &%  %   TTm†@1@ LPO? %  % ( % ( &%  < 6  % &% (    % ( '% &%  < 6    % ( '% ( % % | O %  '% ( &% | O % | O  TT*9m†@1@| O LPN8 % %  % ( % (  O %  O  TT*9m†@1@ O LPH8 % %  a %  a  TT0;m†@1@ a LP2& % % &%  6   % &% (   a  % ( '% % %  %  '% ( &%  %   TTm†@1@ LPO? % %  % ( % ( J% J TTm†@1@JLPH8 % &% )6 6) % &% (  J % ( '%  % ( % &%  |6 6  % &% (  J % ( '%  % ( % &%  |6  % &% (  J % ( '%  % ( % % % V0:0D:% % B6% % V,DD% %  % %  J % '%  % ( % &% 626F CDIFVjCD0100ChemDraw 8.0.33&U@"=Z"    :;<   a  3xx# $$$$D $ArialTimes New RomanxXX0pg{XX0d'`>33 $$s3&UNSϳu3&UNSϳN 3&UNSϳO fXttV  7P N  7Q 1O~"W  7R o*i  +;f=f;8m/An atom in this label has an invalid valence. # aNS Rr  T t}h U PJ V s1+ W dU X d7q Y /C +/bbVb//An atom in this label has an invalid valence. # aNZ <y 7[ =`  7\ |s !7] {{ ' N+{{9*{vl{ #a Pt^ 7$ h *+7 Blj7 Yj7 Bl/An atom in this label has an invalid valence. #aaNH2_ dO ++7UQOjwOjwO/An atom in this label has an invalid valence. #aaNH2` U/h .7a xfUNj 07b 7%hr 27c gBM 37d .S* 47e H" 501 f ~Թ+=r 601 g C``i 77h \.O P  m i i \.P Q j \.Q R  i x k k \.R S l \.S T  k n r m m \.O T n \.S U o \.U V  n s w p p \.V W q \.W X  p } | r r \.T X s "\.U Y t #\.Y Z  s y u u $\.Z [ v %\.[ \  u w w &\.V \ x (\.R ] y )\.] Y z ,\.] ^ { -\._ ] | /\.X ` } 1\.W a ~ 8\.b c  9\.c d  :\.d e  ;\.e f  <\.f g  =\.g b  >\.^ f   ?\._ e  -rk>"-rk>" -rk>"-rk>" CdBX k +dTvdTʠ[ /An atom in this label has an invalid valence. # a S dBX l +dT$dTh%ʠ[ /An atom in tF CDIFhis label has an invalid valence. # a S d m7 Ѥl n7 Jүէv o7 vw p+{czs≿zs{c z/An atom in this label has an invalid valence. # aN ? q /  r + y {ߙ z  { -K | Ƕw +EtEtEx{/An atom in this label has an invalid valence. # aN -[w 7 | 7 @~J 7 [>h  N+ϣ[d6[di[n #a Pt ?~ 7 j 7= 7Ѥ% 7Jү́6 7v6 +{c~2≿~2{c9/An atom in this label has an invalid valence. # aN$ s $ 'J  s !- "ǶL5 +118/An atom in this label has an invalid valence. # aN#-[`5 7$# 7%@# 7&GD  N+ϣGD6G?iGI #a Pt'? 7( 7I7K O" ] +7wFsYl~sY` #aaNHQ7>n S"q 7U7֤ 7W"ס Y7  ["= +7UUĆU" # aO]"ǥ: _7r) +7%j%4 - #aaNHa"? 7c7  e" +7UĆ # aOvǥ: +7zǽ6-rǽ6q {> # aO`{  +7,h$$ײ$ #aaOH`{>n +7,hVjVjײ#r # aOvס +7z?-r?-|ϰ #aaNH2`{  +7,h""ײ" #aaOHK 7K $ ] +7c(sY sY~1` #aaNH>n $q 7֤ 7$ס   $= +7c(U U!)" # aO$ǥ: r) F CDIF+7א%=%_9 - #aaNH$? 7  $ +7c( !) # aO8ǥ: +7<ǽ64ǽ67<> # aOY  +7% $2$V $ #aaOHY>n +7% Vj2VjV #r # aO8ס +7<?4?>ϰ #aaNH2Y  +7% "2"V " #aaOHK 7 s     t   u      v   w      x   }   ~                                                          )  .**  +  *,9,  -  /,.3.  /  0  4/181   2  ! <13;3 ! 4 " 5 "# :466 #$ 7 $% 688 % 9 & : &" ; !' <  ( B & F & P OI R QO T SQ V US X WU Z YW \ [Y^ ]I ` _] b a_ d ca f ec ] c  Q W  Y     I                     F CDIF              7 @"=Z<0"P @ ` A01@ A C+7d@"=@"= D # aO^4P E+7*8L0L8[ #aaOH0"o G7#R_U I+7RQ7 RQX # aHfpj K+7{Sfy fyhy #aaNH2 M70"Y O Q+7d # aO^4Y S+7*8Y0Y*8U #aaNHhCXK U01  aF7(y W+7-3;uY3u-3;| # aH %Nuw Y7 F?i [ +7 J?m3B(_AXJ?m #a aSHJIH ]jC7C _+7?7&77Gjޞ #aaNHJnVH a+7[Z`}R`Z- # aOJIy= c7"nC1 e%JI$ g+7M<}E<M< #aaOH(<61 i+7:Io2I; # aO B\.  D\. F\.  H\.  J\.  L\.  N\.  P\.  R\. T\.  V\.   X\.   Z\.   \\.   ^\.  `\.  b\.! d\. $ f\." ' h\."% * j\."(;@@    <,|t|    ?@ s  ;F DMHCGRTS`~  x (XPa5    J@ACJEG.I4PK M "O XQ "SD iUQ wW UYz?[ E]D !_ Ea cD egG ihkNl m n EoEMpq6ryG z|!V{!M|V][ $,"C"   BE96G|19V)+yb,C  D   L  ! #T  <   5 H ) ) H  )! [DL !#T<5oo !\  [%  @ H / H J   6 m  l e ,   B! m ' ^-* O +#. 02 V3 4 R56;7     "#& ',-347 8;<ABCDFJKLNOTUVWY]^_cdjkn oqBDFHJLNP R T V X Z \ ^`bdfhjstuvwx}~  !!""##$$%%&&"!' 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'   '  '   '  '  '   '  '  '  '  '  '  '  ' )< ' )< 0   !S% ~)WEGEX!S% ~)WEmDX!% ED!% tED!% (jED!N% kjE[TDX!% 'uEE!% EdD!% RuEU E!% E?E!% XEA E!% x7E9D!N% +jE<EX!% jEE!% uE1$E!% G7EE!NPt% &aEbEX!% hEkD!% !EE!% f,ED!% 7ED!% BED!N% >AE[TDX!% $7EE!% h,EdD!% 7EU E!% X,E?E!% 4!EA E!% !E9D!N% OAE<EX!% h\AEE!% L6E1$E!% v+EE!NPt% KEbEX!% 4 EkD!% EE!% ~gOEǹD!N%b `'ZEj]DX!% CdEǹD!% $oEj]D!% gzEǹD!% tEj]D!% eEǹD!O% VSEj]DX!% DEj]D!N%b 9EǹDX!% '/Ej]D!% g$EǹD!O% Ej]DX!O% DE8DX!O%b g$E8DX!O% CdE8DX!N%b tE8DX!O%b eE8DX!% POECD!% gOE10E!N%b a'ZE7EX!% CdE10E!% $oE7E!% gzE10E!% tE7E!% eE10E!O% VSE7EX!% DE7E!N%b 9E10EX!% '/E7E!% g$E10E!O% E7EX!O% DE{CEX!O%b g$Es$EX!O% CdEs$EX!N%b tE{CEX!O%b eEs$EX!% POE"E ' (.  '  ' (.  '  ' (.  '  '  '  (.  '   '  (.  '   '   '  (.  '   ' (.  '   '  '  F MDLSK '   '   '  '  ' (.  '  ' (.  '  ' (.  '  '  ' (.  '  ' (.  '  '  ' (.  '  '  (.  '   '!  '!  '"  '#  '!  '!  '%$  '&%  ''&  '('  ')(  '*)  '+* (  ',$  '-,  '.-  '/.  '0/ (  ',1 (  '/2  '&3 (  ')4  '*5  '6  '6$  '87  '98  ':9  ';:  '<;  '=<  '>= (  '?7  '@?  'A@  'BA  'CB (  '?D (  'BE  '9F (  '<G  '=H  'I7  'I  0+!% ,XID5E!% pD 0E!O% "D 0EX!O%b ,XID[@EX!% jD5E!H% MnUDdv)E!N%b D-)EX!% ɞD 0E!% /)D5E!O% vD 0EX!N%b /)D[@EX!% .D!JE!H% sDBE!% ,SDyPE!S%b D~KEX!% 4[DNE!N%b D"JEX!O% 4[DVEX!% DNE!% d}D %JE!O%b -DNEX!O% d}D=BEX '  ' (  '  '  ' )< ' )< '  '  '  (  '   '  )< '  )< '   '   '   '  ' (  '  '  '  ' ( 0  U89d2  0 ~ U C?Ariald+% % ( CONTENTS =1Table, bSummaryInformation(<DocumentSummaryInformation8DVjCD0100ChemDraw 8.0.33&U@"=Z"  @@' :;<   a  3xx# $$$$D $ArialTimes New RomanxXX0pg{XX0dd'`B33 $$s3&UNSϳu3&UNSϳN 3&UNSϳO fXttV  7P N  7Q 1O~"W  7R o*i  +;f=f;8m/An atom in this label has an invalid valence. # aNS Rr  T t}h U PJ V s1+ W dU X d7q Y /C +/bbVb//An atom in this label has an invalid valence. # aNZ <y 7[ =`  7\ |s !7] {{ ' N+{{9*{vl{ #a Pt^ 7$ h *+7 Blj7 Yj7 Bl/An atom in this label has an invalid valence. #aaNH2_ dO ++7UQOjwOjwO/An atom in this label has an invalid valence. #aaNH2` U/h .7a xfUNj 07b 7%hr 27c gBM 37d .S* 47e H" 501 f ~Թ+=r 601 g C``i 77h \.O P  m i i \.P Q j \.Q R  i x k k \.R S l \.S T  k n r m m \.O T n \.S U o \.U V  n s w p p \.V W q \.W X  p } | r r \.T X s "\.U Y t #\.Y Z  s y u u $\.Z [ v %\.[ \  u w w &\.V \ x (\.R ] y )\.] Y z ,\.] ^ { -\._ ] | /\.X ` } 1\.W a ~ 8\.b c  9\.c d  :\.d e  ;\.e f  <\.f g  =\.g b  >\.^ f   ?\._ e  -rk>"-rk>" -rk>"-rk>" CdBX k +dTvdTʠ[ /An atom in this label has an invalid valence. # a S dBX l +dT$dTh%ʠ[ /An atom in this label has an invalid valence. # a S d m7 Ѥl n7 Jүէv o7 vw p+{czs≿zs{c z/An atom in this label has an invalid valence. # aN ? q /  r + y {ߙ z  { -K | Ƕw +EtEtEx{/An atom in this label has an invalid valence. # aN -[w 7 | 7 @~J 7 [>h  N+ϣ[d6[di[n #a Pt ?~ 7 j 7= 7Ѥ% 7Jү́6 7v6 +{c~2≿~2{c9/An atom in this label has an invalid valence. # aN$ s $ 'J  s !- "ǶL5 +118/An atom in this label has an invalid valence. # aN#-[`5 7$# 7%@# 7&GD  N+ϣGD6G?iGI #a Pt'? 7( 7I7K O" ] +7wFsYl~sY` #aaNHQ7>n S"q 7U7֤ 7W"ס Y7  ["= +7UUĆU" # aO]"ǥ: _7r) +7%j%4 - #aaNHa"? 7c7  e" +7UĆ # aOvǥ: +7zǽ6-rǽ6q {> # aO`{  +7,h$$ײ$ #aaOH`{>n +7,hVjVjײ#r # aOvס +7z?-r?-|ϰ #aaNH2`{  +7,h""ײ" #aaOHK 7K $ ] +7c(sY sY~1` #aaNH>n $q 7֤ 7$ס   $= +7c(U U!)" # aO$ǥ: r) +7א%=%_9 - #aaNH$? 7  $ +7c( !) # aO8ǥ: +7<ǽ64ǽ67<> # aOY  +7% $2$V $ #aaOHY>n +7% Vj2VjV #r # aO8ס +7<?4?>ϰ #aaNH2Y  +7% "2"V " #aaOHK 7 s     t   u      v   w      x   }   ~                                                          )  .**  +  *,9,  -  /,.3.  /  0  4/181   2  ! <13;3 ! 4 " 5 "# :466 #$ 7 $% 688 % 9 & : &" ; !' <  ( B & F & P OI R QO T SQ V US X WU Z YW \ [Y^ ]I ` _] b a_ d ca f ec ] c  Q W  Y     I                                   7 @"=Z<0"P @ ` A01@ A C+7d@"=@"= D # aO^4P E+7*8L0L8[ #aaOH0"o G7#R_U I+7RQ7 RQX # aHfpj K+7{Sfy fyhy #aaNH2 M70"Y O Q+7d # aO^4Y S+7*8Y0Y*8U #aaNHhCXK U01  aF7(y W+7-3;uY3u-3;| # aH %Nuw Y7 F?i [ +7 J?m3B(_AXJ?m #a aSHJIH ]jC7C _+7?7&77Gjޞ #aaNHJnVH a+7[Z`}R`Z- # aOJIy= c7"nC1 e%JI$ g+7M<}E<M< #aaOH(<61 i+7:Io2I; # aO B\.  D\. F\.  H\.  J\.  L\.  N\.  P\.  R\. T\.  V\.   X\.   Z\.   \\.   ^\.  `\.  b\.! d\. $ f\." ' h\."% * j\."(;@@    <,|t|    CD s  ;                          ! " # $ % & ' ( ) + - . / 0 1 2 3 4 5 6 7 8 9 : ; < = > ? @ A B C D E F G H I J K L M N O P Q R S T U V W X Y Z [ \ ] OL0КuR[ZBfpnjv<<TVkc.WTbr Ǵ퇲|& b-)@Дxz ^dUC gܢ({`S??e deُ- .N~&cP%؅}qn=?>G+"L tffix$`eŪI <4Cyy~>G?6M:Bv{)6n ;#07lVur,o.Kadcp˗m{WTmMA$5=K)W)Bz<'_?ʽoOG|2t~{3#lg;3y=NSO)pb4XȨqh`^e'\}}*T/ usѼ׽uO7[9F}'-ʥ? 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