Multifunctional Pt(II) Reagents: Covalent Modifications of Pt

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Multifunctional Pt(II) Reagents: Covalent Modifications of Pt Complexes Enable Diverse Structural Variation and In-Cell Detection Jonathan D. White, Michael M. Haley, and Victoria J. DeRose* Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon 97403-1253, United States CONSPECTUS: To enhance the functionality of Pt-based reagents, several strategies have been developed that utilize Pt compounds modified with small, reactive handles. This Account encapsulates work done by us and other groups regarding the use of Pt(II) compounds with reactive handles for subsequent elaboration with fluorophores or other functional moieties. Described strategies include the incorporation of substituents for well-known condensation or nucleophilic displacement-type reactions and their use, for example, to tether spectroscopic handles to Pt reagents for in vivo investigation. Other chief uses of displacement-type reactions have included tethering various small molecules exhibiting pharmacological activity directly to Pt, thus adding synergistic effects. Click chemistry-based ligation techniques have also been applied, primarily with azide- and alkyneappended Pt complexes. Orthogonally reactive click chemistry reactions have proven invaluable when more traditional nucleophilic displacement reactions induce side-reactivity with the Pt center or when systematic functionalization of a larger number of Pt complexes is desired. Additionally, a diverse assortment of Pt−fluorophore conjugates have been tethered via click chemistry conjugation. In addition to providing a convenient synthetic path for diversifying Pt compounds, the use of click-capable Pt complexes has proved a powerful strategy for postbinding covalent modification and detection with fluorescent probes. This strategy bypasses undesirable influences of the fluorophore camouflaged as reactivity due to Pt that may be present when detecting preattached Pt−fluorophore conjugates. Using postbinding strategies, Pt reagent distributions in HeLa and lung carcinoma (NCI-H460) cell cultures were observed with two different azide-modified Pt compounds, a monofunctional Pt(II)−acridine type and a difunctional Pt(II)-neutral complex. In addition, cellular distribution was observed with an alkyne-appended difunctional Pt(II)neutral complex analogous in structure to the aforementioned difunctional azide-Pt(II) reagent. In all cases, significant accumulation of Pt in the nucleolus of cells was observed, in addition to broader localization in the nucleus and cytoplasm of the cell. Using the same strategy of postbinding click modification with fluorescent probes, Pt adducts were detected and roughly quantified on rRNA and tRNA from Pt-treated Saccharomyces cerevisiae; rRNA adducts were found to be relatively long-lived and not targeted for immediate degradation. Finally, the utility and feasibility of the alkyne-appended Pt(II) compound has been further demonstrated with a turn-on fluorophore, dansyl azide, in fluorescent detection of DNA in vitro. In all, these modifications utilizing reactive handles have allowed for the diversification of new Pt reagents, as well as providing cellular localization information on the modified Pt compounds.

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INTRODUCTION AND SCOPE Since the discovery of their biological activity in the mid-1960s, platinum(II)-based therapeutics have been a mainstay of anticancer drug treatments. Today, over half of anticancer regimes include at least one of three FDA-approved Pt(II)based drugs: cisplatin, carboplatin, and oxaliplatin (Figure 1).1 Despite their success against different cancers, Pt therapeutics are not universally effective. Treatment with these drugs induces undesirable side-effects. Also, certain cancers have resistance to Pt-based therapies, either intrinsically or developing during prolonged treatment. The reactivity of these Pt reagents involves the loss of one or more labile leaving group ligands and replacement with water ligands, followed by facile displacement of water and binding of Pt to cellular nucleophiles.2 DNA−Pt adducts have been the best-characterized, because Pt forms stable adducts with neighboring N7 nitrogen atoms of purine nucleobases, interrupting normal transcription processes of the cell and causing downstream apoptotic signaling.3,4 The nonleaving © XXXX American Chemical Society

nitrogen-donor ligands are not displaced, but play an important role in determining the nature of the Pt−DNA adduct, which in turn governs the cross-resistivity and sensitivity of different cancer cell types toward Pt.5 In one characterized pathway, failure to repair DNA−Pt lesions results in recruitment of the high-mobility group (HMG) proteins, which initiates p53 and eventual cell-cycle arrest and apoptosis.6 Although binding to DNA has been a major research focus, the interactions of Pt compounds are complex and directed toward a diverse range of biological nucleophiles (Figure 2). In addition to DNA, Pt is known to bind cellular proteins, RNAs, and small molecules such as glutathione through sulfur- or nitrogen-donor moieties.7−10 A comprehensive molecular-level description of these interactions of Pt with alternative targets is unavailable, and their consequences, such as those affecting cellular Received: July 2, 2015

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DOI: 10.1021/acs.accounts.5b00322 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Three FDA-approved Pt-based anticancer therapeutics (left). The halide or O-donor ligands are eventually displaced by biomolecules, while the N-donor ligands are generally retained on the final Pt−biomolecule adduct (right).

Figure 2. Summary of the cellular reactivity of Pt(II)-based therapeutics. (1) Pt enters the cell through both active transport and passive diffusion modes.11 Cu transport proteins, organic cation transporters, and endocytosis have all been implicated in active transport. (2) Lower intracellular chloride concentrations (∼4 vs ∼100 mM extracellular) favor displacement of chloride by aqua ligands, forming cationic exchange-labile Pt species. Binding of Pt to small anions such as carbonate and phosphate may also occur, which would affect rates of subsequent Pt substitution.12,13 (3) Pt preferentially binds guanine and adenine residues of nuclear DNA forming exchange-inert lesions. A 1,2-intrastrand cross-link of cisplatin on DNA is depicted (inset, PDB 3LPV).14 These lesions interrupt normal transcription processes of the cell, inducing cell-cycle arrest and apoptosis. (4) Alternative targets of Pt include the N- and S-donor moieties of cellular proteins, RNAs, and small molecules such as glutathione (GSH).7−10 Binding to these targets and their consequences relating to influx and efflux of Pt, cytotoxicity, resistance mechanisms, and alternative apoptotic signals, among others, are poorly understood. (5) Pt may be shuttled out of the cell bound to small molecules such as glutathione via GS−X pumps or other modes.11

reagents with fluorophores. Others have desired facile methods to conjugate Pt drugs to different groups for purposes such as improved efficacy, achieving synergistic therapeutic effects, and optimized transport and delivery. Modulation of the ligands to incorporate bioactive groups is a difficult task due to the necessity of retaining essential features of the Pt complexes, such as their cis geometry and number of inert and labile ligands.3 Incorporation of additional groups is usually performed via alkylation of the ammine ligands or derivatization of other N-donor ligands of Pt drugs, followed by coordination to Pt using established methods.26 Integration of modified ligands becomes increasingly difficult when the desired product contains nonequivalent or mixed-ammine/ amine groups, as it is difficult to control the degree of substitution on Pt. In addition, undesired side reactivity due to the appended groups may produce small amounts of impurities, which are often difficult to remove. Once methods of synthesis and purification have been established for a particular complex, such techniques are frequently nontransferrable and unfeasible with other, even similar systems; thus, creation of even the smallest library of modified Pt drug compounds may be a tedious and laborious task as synthetic methods involving Pt are rarely broadly applicable. It is the desire and theme of endowing Pt reagents and drug candidates with additional functionality that is the focus of this Account. We hope to stimulate use of these strategies in future work seeking to both functionalize reagents and detect or study Pt in biologically relevant systems. Direct covalent modification of the ligand(s) bound to Pt is a possible route, one commonly employed to attach fluorescent reporters directly to Pt. To

transport, cytotoxicity, Pt resistance, and alternative apoptotic signals, are poorly understood.2−4 Despite the continuing need to track and observe Pt compounds in biologically relevant systems, the minimal complexity of the native drugs presents a challenge. Once bound to a cellular target, the Pt center and coordination sphere are relatively unreactive, presenting few options for postbinding functionalization and detection. In one approach, the unique properties of Pt itself have been utilized with methods that include detection of radioactive Pt isotopes,15,16 X-ray fluorescence and electron microscopy,17,18 X-ray absorption near-edge spectroscopy,19 NMR spectroscopy,20 and mass spectrometry (MS).21 As previously reviewed, many important attributes of Pt drug activity have been determined through these methods.22,23 Despite the diversity and early success of Pt-based detection methods, significant disadvantages include their inability to observe the encompassing ligand environment of the Pt compound and their incompatibility with live-cell systems. As another approach to identify proteins that bind DNA−Pt adducts, modified constructs have been used as “bait” to capture proteins from cell extract.4 In one recent example,24 a poly(His) peptide tag allowed for facile pull-down and subsequent MS identification of the “captured” proteins. With this approach, some differences in the recruited proteins were found for different Pt anticancer therapeutics.25 This strategy represents one method of functionalizing Pt(II) compounds for mechanistic investigations. As another approach for in vivo detection of Pt compounds, significant effort has been put toward functionalizing Pt B


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Figure 3. Examples of nucleophilic displacement reactions to functionalize Pt(II) and Pt(IV) complexes. Both outer-sphere esterification reactions (A)28 and peptide couplings (B)29 have been demonstrated on Pt(II), as well as reactions involving the inner-sphere ligands of Pt(IV) complexes (C).27 BOP-Cl = bis(2-oxo-3-oxazolidinyl)phosphinic chloride. DIPEA = N,N-diisopropylethylamine.

Figure 4. Examples of click chemistry reactions used to functionalize Pt complexes. The Cu-catalyzed azide−alkyne cycloaddition reaction was used to generate small libraries of Pt−diazenecarboxamide conjugates for improved drug sensitivity of Pt (D).34 The Pt(IV) prodrug of oxaliplatin was appended via Cu-catalyzed click with a fluorescent quinone moiety (E).35 Strain-promoted Cu-free click chemistry was used to functionalize Pt(IV) cisplatin prodrugs (F).36 Metal-mediated amine-to-nitrile click reactions have been utilized by Bierbach to synthesize a library of Pt−acridine reagents following the general formula of 1 (G).37

C


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Figure 5. Examples of fluorophore-tethered Pt complexes used to observe Pt cellular distribution in cells. Reedijk investigated diacetylfluoresceintethered ethylenediamine Pt(II), 2, in U2-OS cancer cells, observing nuclear accumulation after 2−3 h and cytoplasmic accumulation after several hours.38 Howell showed colocalization of 3 in the vesicular structures of the lysosomal, Golgi, and secretory compartments of cells.39 Observation of the anthraquinone-containing polynuclear 4 implicated glutathione-independent resistance mechanisms in cisplatin-resistant human ovarian carcinoma cells.40 BODIPY conjugate 5 was used to image localization and DNA damage in live-animal xenograph cancer mouse models by Weissleder.29 Pretethered monofunctional Pt−fluorophores incorporating a nitrobenzene group, such as 6, were used to observe Pt localization in HeLa cells42 and zebrafish larva43 by Guo. The pretethered 7 was used for Pt−acridine localization in lung cancer cells by Bierbach.41 Molecular charges are omitted for clarity.

sphere.30 Octahedral Pt(IV) compounds have been investigated for their anticancer activity due to their unique propensity to be reduced in vivo to the corresponding Pt(II) congeners with loss of two axial ligands.31 Pt(IV) prodrugs bearing biologically relevant carboxylate axial ligands have been prepared.32 Synthetic methods involving the formation of Pt(IV) complexes bearing appropriately reactive groups and their further modifications via nucleophilic substitution reactions have been reviewed recently.26 In all cases, care must be taken in selecting the proper complementary conjugates to append onto Pt complexes such that they do not react directly with the Pt center. One drawback of using hydroxyl, amino, and carboxylate functional groups for further decoration of Pt compounds is that the subsequent attachment of functional groups cannot be performed in biological environments due to obvious nonselective cross-reactivity. Thus, for example, there is no option to add a fluorophore or biotin for subsequent detection after the Pt compound has entered a cell and bound its biological partner. To improve the selectivity of conjugation reactions, including for use in biological systems in situ, click chemistry has also been pursued for facile conjugation of bioorthogonal substituents using click-modified Pt compounds.

create more general synthetic approaches, small, minimally invasive reactive handles have been incorporated onto Pt, allowing for facile covalent modification with complementary reactive conjugates. This latter approach has been used to detect Pt in-cell using postbinding covalent modifications with click chemistry, utilizing complementary click-modified fluorescent probes. Both pre- and postbinding approaches of modifying Pt compounds and their advantages and disadvantages are assessed below.

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NUCLEOPHILIC SUBSTITUTION REACTIONS FOR FUNCTIONALIZATION OF Pt COORDINATION COMPOUNDS Nucleophilic substitution reactions including esterification and peptide coupling have been used on Pt complexes whose ligands are functionalized with the appropriate reactive handles. Such handles have included alcohols and amines, as well as carboxylic acids incorporated into Pt ligands such that they remain available for further reaction.26 In addition, the sufficiently nucleophilic Pt-bound hydroxide ligand(s) of some Pt(IV) complexes have been shown to undergo acetylation reactions with anhydrides, pyrocarbonates, and isocyanates to form the dicarboxylates, dicarbonates, or dicarbamate Pt(IV) species, respectively.27 All of these reactions allow further derivatization of Pt species with a broad range of appended groups; examples include nonsteroidal anti-inflammatory compounds to overcome Pt resistance,28 fluorescent probes,29 and reactive thioether moieties27 (Figure 3). Pt(IV) compounds have also been modified via condensation reactions using moieties in the secondary coordination

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CLICK CHEMISTRY FOR FUNCTIONALIZATION OF Pt COMPOUNDS Click chemistry has been used for the diverse functionalization of Pt compounds using selectively modified Pt complexes and their partner conjugates. Click chemistry, as defined by Sharpless and colleagues, refers to a set of reactions designed to build molecules by joining small units together. Click D


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Figure 6. Strategies incorporating reactive handles onto Pt for atom-based detection, nucleophilic-based substitution and functionalization, clickchemistry-based functionalization, and pretethered fluorophore-based detection methods.

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FLUOROPHORE-TETHERED COMPLEXES FOR IN VIVO DETECTION OF Pt COMPOUNDS The added functional utility of a fluorescent probe on Pt has provided much information about the properties of Pt compounds in biological environments via cellular localization studies. Different synthetic strategies have been employed to covalently attach fluorophores to Pt complexes, most commonly as fluorophore-appended ethylenediamine ligands. Monofunctional Pt(II) compounds with only one available labile chloride ligand for displacement have also been tethered to fluorophores. Examples are highlighted in Figure 5, along with results from cellular localization studies performed with these conjugates.29,38−43 Other recent reviews provide additional history of the use of pretethered Pt−fluorophore conjugates.22,23,44 Indeed, the added functional utility of appended fluorescent probes has provided the capability to track and observe the influx, distribution, and efflux of Pt−fluorophore species in cells. It is important to consider, however, that studies using appended fluorescent probes are only detecting fluorescent emission of the probe and not necessarily the presence of intact drug. The assumption that the probe remains attached to the complex throughout detection may not always hold true.45 Importantly, the physical properties of the bulky organic fluorophores and their interactions in cellular environments may not mimic those of small, neutral or positively charged Pt drugs. Fluorophores bound to Pt are expected to influence the types of covalent, intercalative, hydrophobic, and electrostatic interactions that guide localization of the Pt compound.2,46 In a recent example of ligands influencing recognition properties, Chow and colleagues demonstrated altered binding specificities of Pt toward 16S rRNA (in vitro) that depended on the identity of small, primary-amine-bearing amino acid ligands coordinated to Pt.47 It is desirable, therefore, to incorporate increasingly less

reactions are distinguished as being modular and broad in scope, high-yielding, and orthogonal.33 Advantages of using the azide−alkyne cycloaddition click reaction to functionalize Pt complexes include its selective reactivity and its usefulness when more traditional nucleophilic displacement reactions induce side reactivity with the Pt center or when diverse functionalization of a larger number of desired Pt complexes is synthetically demanding. A variety of click chemistry reactions have been used to synthesize functionalized Pt compounds (Figure 4). Cu(I)-catalyzed azide−alkyne cycloaddition reactions were used by Urankar and colleagues to synthesize small libraries of carboplatin derivatives conjugated to bioactive diazenecarboxamide carrier ligands.34 Zhang et al. used azide-appended Pt(IV) prodrugs with catalytic CuI to react with a (propargylamino)anthraquinone group, demonstrating facile Pt(IV) functionalization incorporating asymmetric, or mixed, axial groups.35 Cu-free, strain-promoted azide−alkyne cycloaddition has been demonstrated by Dhar and colleagues with Pt(IV) cisplatin prodrugs as a platform to conjugate Pt to the far-red Cy5.5 dye.36 The pretethered fluorophore attached to Pt(IV) was used to visualize the cellular uptake of Pt in live prostate cancer cells. Interestingly, in control experiments, incorporation of the organo-azide moiety onto the Pt(IV) cisplatin prodrug was found to increase overall cellular toxicity for undetermined reasons. Bierbach and colleagues have demonstrated the utility of metal-mediated amine-to-nitrile addition,37 another type of click reaction (Figure 4G). This conjugation strategy was used to generate Pt−acridine agents for use as cytotoxic “warheads” in targeted multifunctional therapies, synthesizing a library of Pt−acridine compounds containing hydroxyl, carboxylic acid, and azide moieties, the latter providing a second handle for further modification (vide infra). E


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the presence of terminal alkyne groups appended onto Pt through short organic linkers facilitates the possibility of η1- or η2-coordination of the alkyne to Pt, an undesirable side reaction impeding click ligation.54 Notwithstanding, a handful of alkyneappended complexes have been prepared and shown to be stable toward alkyne−Pt side reactions.55−58 Pt(II) complexes that incorporate accessible alkyne moieties and also have labile halide or O-donor ligands remain rare, however. Alkyne 11 (Figure 6)52 has been successfully used in postbinding labeling reactions with fluorogenic probes. Alkyne-appended Pt complexes may provide advantages over their azide-functionalized counterparts, including improved lipophilicity and access to complementary azide-modified partner reagents such as turn-on azide-appended fluorophores. Pt−acridine anticancer compounds such as 1 (Figure 4) are a potent class of antitumor agents, exhibiting cytotoxicities up to 500 times greater than cisplatin in non-small-cell lung cancers.59 The compounds derive their cytotoxicity from combining the intercalative capability of the acridine group with the covalent modification of Pt−DNA lesions.60 To obtain accurate cellular localization for this Pt−acridine family, Bierbach synthesized analogue 8 (Figure 7) containing the azide handle for use with fluorescent postlabeling via click chemistry. Compound 8 exhibits somewhat higher IC50 values37 but retains the submicromolar inhibitory effects measured in the most potent Pt−acridine hybrid compounds (lung cancer cell culture). Human non-small-cell lung carcinoma (NCI-H460) cells were treated with 8, fixed, permeabilized, and then incubated in a Cu-containing buffer with the alkyne-functionalized bright-green Alexa Fluor 488 dye.48 Increased fluorescence intensity was localized more strongly in the nucleus of cells. Approximately 50% greater nucleolar localization relative to the surrounding chromatin was observed in cells during the interphase cell cycle, while cells undergoing mitosis exhibited intense fluorescent labeling of condensed chromatin. Additionally, colocalization studies with pretethered 7 (Figure 5) showed a different distribution pattern in cells, with significantly higher fluorescence observed in the chromatin (relative to that observed in the nucleolus and cytosol). The difference in localization with 7 vs post-treatment modification of 1 was attributed to the added bulk of the DIBO moiety in 7 affecting target binding of the Pt complex and its ability to associate and form adducts with double-stranded DNA of the chromatin. In all, these studies validated the use of postbinding fluorescent click labeling to observe Pt accumulation in cells. The imaging results also supported the conclusion that nuclear DNA damage by 1 is responsible for the primary commencement of S-phase arrest and apoptosis (in NCI-H460 cells). Our group has previously investigated the cellular reactivity of cisplatin toward RNA, using S. cerevisiae as a model organism. ICP-MS of extracted RNA in cells treated with cisplatin revealed approximately 4−20-fold greater accumulation of Pt on total RNA versus DNA.21 To further study the target binding of small, difunctional Pt(II) compounds, we synthesized azide 9,49 a derivative of the sterically hindered drug candidate picoplatin, for postbinding fluorescent labeling. Steric hindrance provided by the 2-methyl group of picoplatin and 9 slows the kinetics of Pt substitution reactions and is an important effector of biological activity.61 After treatment with 9, dose-dependent fluorescent postlabeling of S. cerevisiae rRNA and tRNA (Figure 9) revealed significant labeling of all rRNA bands.50 To our knowledge, it is also the first demonstration of

invasive yet reactive handles in order to observe Pt compounds not through preconjugation of the fluorescent probes but through postbinding covalent modification. To this end, the azide−alkyne cycloaddition click reaction has been used to perform conjugation reactions on Pt post-treatment, allowing for more accurate localization and characterization of Pt-bound cellular targets. Strategies incorporating the above-discussed reactive handles, including atom-based detection methods, nucleophilic displacement reactions, click chemistry reactions, and pretethered fluorescent probes are summarized in Figure 6.

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CLICK CHEMISTRY FOR Pt DETECTION VIA POSTBINDING BIOORTHOGONAL LIGATION To broadly study cellular Pt interactions, the DeRose and Bierbach groups have functionalized Pt compounds with bioorthogonally reactive handles for use in postbinding modification reactions. Chosen due to the biological compatibility, exquisite selectivity, and high yields of the azide−alkyne cycloaddition click reaction, several Pt compounds have recently been synthesized containing azide or alkyne handles for functionalization with fluorescent probes (Figure 7) once bound to a biomolecule target.41,48−53

Figure 7. Click-functionalized Pt compounds used in postbinding modification reactions to observe Pt via conjugation with fluorescent probes. Bierbach and colleagues have synthesized and investigated the cellular (NCI-H460) distribution of the Pt−acridine compound 8.41,48 DeRose et al. have synthesized picoplatin derivative 949 and identified cellular RNA targets of Saccharomyces cerevisiae via ex vivo click labeling.50 Azide 10 was used to further characterize cellular RNA targets of S. cerevisiae.51 Alkyne 11 was investigated in postbinding reactions with the turn-on fluorophore dansyl azide.52 Both 11 and azide 12 were used to investigate Pt cellular distribution in HeLa cells via click labeling with complementary rhodamine fluorophores.53

This strategy takes advantage of bioorthogonal click reactions not simply for the preconjugation of fluorescent probes but for conjugation to Pt-bound targets in-cell or extracted from cells (Figure 8). With the fluorophore introduced after the Pt is bound to its cellular targets, this strategy avoids unwanted side reactivity or directing effects from bulky organic fluorophores in biological systems. Aliphatic azide groups may be installed on amine-containing Pt ligands relatively easily via nucleophilic displacement with N3−, and these azide-appended ligands may be coordinated to Pt(II) free of side reactions between R−N3 and Pt. In contrast, F


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Figure 8. Postbinding covalent modification workflow used by Bierbach and DeRose for the investigation of biological Pt targets using clickchemistry-enabled Pt derivatives. In addition to fluorescent labeling of Pt targets, potential applications of postbinding click modifications include Pt−biomolecule pull-down followed by high-throughput sequencing or proteomics to identify nucleic acid or protein targets, respectively.

Figure 9. (a) Ex vivo fluorescent analysis of postlabeled 9-bound rRNA and tRNA purified from Pt-treated S. cerevisiae. (b) Dose-dependent fluorescent labeling of cellular RNA is observed in the middle and right lanes, indicating rRNA and tRNA are targets of Pt in vivo.50 Cells were treated with 0, 250, or 500 μM 9 for 6 h. Harvested RNAs were then reacted with an excess of Alexa Fluor 488 DIBO alkyne for 18 h at 37 °C and subjected to dPAGE.

in vivo Pt accumulation in tRNA. Of note, relatively high Pt concentrations were used to combat the low sensitivity of detecting in-gel fluorescence (Figures 9 and 10, vide infra) and the slower reactivity of the methyl-shielded 9 in S. cerevisiae (Figure 9). In addition to 9, sterically unhindered 10 first synthesized by Urankar was used to probe the longevity of Pt−rRNA adducts in S. cerevisiae.51 To determine whether Pt−rRNA adducts were quickly degraded or long-lived, cells were treated with 10 and then resuspended in fresh media. RNA was extracted at varying times, and Pt adducts were detected following click labeling with Alexa Fluor 488 (Figure 10). Cells resuspended in drugfree media showed growth similar to nontreated control cells, consistent with the relatively lower toxicity of diamine-chelatecontaining Pt compounds and in contrast to treatment with cisplatin.62 Consequently, higher treatment concentrations of 10 could be used to label RNA in S. cerevisiae, improving the yield of labeling. In these experiments, the utility of the alkynereactive azide handle to identify bound Pt species is clearly demonstrated and further validates the utility of click-reactive handles in postbinding modification reactions.

Figure 10. Fluorescent labeling via click chemistry of 25S and 18S rRNA extracted from 10-treated S. cerevisiae. S. cerevisiae was treated with 0 or 250 μM 10 for 6 h and resuspended in drug-free media for 0−120 min. Extracted RNA was subsequently treated with Alexa Fluor 488 DIBO alkyne (16 h, 37 °C). No significant loss of fluorescent signal is observed up to 2 h after Pt treatment, indicating that these Pt adducts are not immediately targeted for degradation.51

We have also demonstrated the utility of alkyne-appended complex 11 in click labeling of biomolecules, visualized in-gel.52 Alkyne 11 was used in a Cu-catalyzed azide−alkyne cycloaddition click reaction with the “turn-on” fluorogenic azidecontaining dansyl azide, with no deleterious intra- and G


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Figure 11. Accumulation of 11 and 12 in HeLa cells is visualized with confocal fluorescence microscopy after fixation and click to a rhodamine− alkyne or −azide fluorophore. HeLa cells were treated with 11 or 12 (25 μM, 3 h), then fixed, permeabilized, and reacted with the complementary fluorophore under click conditions (CuSO4 and ascorbate, panels b and d). No labeling is observed in Cu-free controls (panels f and h). Panel j shows comparable fluorescence localization in HeLa cells after treatment with only 5 μM 12, with the comparison of fluorescence and DAPI (panel k) revealing significant accumulation of Pt in the nucleolus. Accumulation in the nucleus and cytoplasm is also apparent. Nucleolar accumulation is consistent with the post-treatment fluorescent labeling by Bierbach,48 indicating potential similarities in the reactivity and targeting of monofunctional Pt−acridine compounds and the difunctional, neutral Pt complexes. Figure adapted from ref 53.

targets and to understand the downstream consequences of their platination. Future work may also include Cu-free click fluorescent ligation for imaging in live cells64 and highthroughput identification of targets isolated via click ligation. While the Pt compounds used in these postbinding click experiments maintain the difunctional cis-diamine ligand geometry of cisplatin, future appropriate modifications to the current FDA-approved Pt(II) compounds, as well as other metallocompounds with appropriate exchange-inert ligands, are promising.

intermolecular side reactions between the terminal alkyne moiety and Pt. In addition to in-gel visualization of postlabeled Pt cellular adducts, we have pursued the use of click-functionalized Pt(II) reagents in cellular localization studies. Localization of Pt adducts of 11 and 12 in HeLa cell culture using post-treatment click fluorescent labeling, observed with confocal microscopy (Figure 11), is successful at Pt treatment concentrations as low as 5 μM.53 The high levels of Pt accumulation in the nucleoli after only 3 h is consistent with both nuclear DNA as a known target of Pt therapeutics and the high levels of Pt−rRNA adducts observed by ICP-MS and postbinding fluorescence labeling in S. cerevisiae.21,50 Because the nucleolus is the site of rRNA synthesis and ribosome assembly, Pt binding to these nuclear RNA targets may contribute to a ribotoxic stress response and is hypothesized to contribute to Pt cellular toxicity,63 and specific platination sites of rRNA have been identified in vitro and in vivo.50 Proteins of the ribosome and other complexes are also potential nucleolar targets of Pt. Interestingly, azide 12 consistently displayed more proficient labeling in cells (see Figure 11, panel b vs d), but with an indistinguishable localization compared with the alkyne 11. Current efforts are underway to determine the extent of Pt binding to nucleolar

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CONCLUDING REMARKS While in its early stages as a new method used to study Pt reagents, postbinding click ligation using azide- and alkyneappended derivatives has already revealed important information regarding target binding and distribution in cells (Figure 12). Advantages of the method include the minimization of structural perturbations to Pt and the commercial availability of click-functionalized partner reagents (e.g., fluorescent probes, biotin), leading to diverse conjugation possibilities with Pt. Disadvantages include the challenges in synthetically modifying Pt to contain the azide- or alkyne-bearing handles and the need to monitor and optimize the click reaction itself. As new H


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Figure 12. Advantages and disadvantages of using click chemistry for postbinding bioorthogonal ligation, including information already revealed utilizing this strategy for detection of Pt.

reagents functionalized with reactive handles are synthesized and new ligation schemes employed, these modified Pt complexes will prove an invaluable tool in further elucidating mechanisms of Pt biological activity.

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University. In 1995, DeRose joined the faculty at Texas A&M University, where she began a program investigating the metallobiochemistry of RNA; she moved to Oregon in 2006 and is currently Professor of Chemistry and Biochemistry.

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AUTHOR INFORMATION

Corresponding Author

REFERENCES

(1) Harper, B. W.; Krause-Heuer, A. M.; Grant, M. P.; Manohar, M.; Garbutcheon-Singh, K. B.; Aldrich-Wright, J. R. Advances in Platinum Chemotherapeutics. Chem. - Eur. J. 2010, 16, 7064−7077. (2) Wexselblatt, E.; Yavin, E.; Gibson, D. Cellular interactions of platinum drugs. Inorg. Chim. Acta 2012, 393, 75−83. (3) Wang, D.; Lippard, S. J. Cellular processing of platinum anticancer drugs. Nat. Rev. Drug Discovery 2005, 4, 307−320. (4) Jamieson, E. R.; Lippard, S. J. Structure, Recognition, and Processing of Cisplatin−DNA Adducts. Chem. Rev. 1999, 99, 2467− 2498. (5) Rixe, O.; Ortuzar, W.; Alvarez, M.; Parker, R.; Reed, E.; Paull, K.; Fojo, T. Oxaliplatin, tetraplatin, cisplatin, and carboplatin: Spectrum of activity in drug-resistant cell lines and in the cell lines of the national cancer institute’s anticancer drug screen panel. Biochem. Pharmacol. 1996, 52, 1855−1865. (6) Arnesano, F.; Natile, G. Mechanistic insight into the cellular uptake and processing of cisplatin 30 years after its approval by FDA. Coord. Chem. Rev. 2009, 253, 2070−2081. (7) Casini, A.; Reedijk, J. Interactions of anticancer Pt compounds with proteins: an overlooked topic in medicinal inorganic chemistry? Chem. Sci. 2012, 3, 3135−3144. (8) Guggenheim, E. R.; Xu, D.; Zhang, C. X.; Chang, P. V.; Lippard, S. J. Photoaffinity Isolation and Identification of Proteins in Cancer Cell Extracts that Bind to Platinum-Modified DNA. ChemBioChem 2009, 10, 141−157. (9) Yu, F.; Megyesi, J.; Price, P. M. Cytoplasmic initiation of cisplatin cytotoxicity. Am. J. Physiol. Renal. Physiol. 2008, 295, F44−F52. (10) Mezencev, R. Interactions of cisplatin with non-DNA targets and their influence on anticancer activity and drug toxicity: the complex world of the platinum complex. Curr. Cancer Drug Targets 2015, 14, 794−816. (11) Arnesano, F.; Losacco, M.; Natile, G. An Updated View of Cisplatin Transport. Eur. J. Inorg. Chem. 2013, 2013, 2701−2711.

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

The authors thank the National Science Foundation (Grant CHE-1413677 to V.J.D.) for funding. Notes

The authors declare no competing financial interest. Biographies Jonathan D. White received his B.S. degree from George Fox University in 2010. He completed his Ph.D. degree in the DeRose and Haley laboratories at the University of Oregon in June 2015 and is currently a postdoctoral researcher at OHSU in the laboratory of Prof. Kimberly Beatty. His general interests include chemical biology, bioinorganic chemistry, and the chemistry of cancer. Michael M. Haley received his B.A. (1987) and Ph.D. (1991) degrees from Rice University and conducted postdoctoral research at the University of California at Berkeley. In 1993, he joined the faculty at the University of Oregon, where he is currently the Richard M. and Patricia H. Noyes Professor of Chemistry. His current research focuses on indenofluorenes, phenylacetylene anion receptors, and other novel carbon-rich systems. Victoria J. DeRose received her B.S. degree from the University of Chicago (1983) and Ph.D. degree from the University of California at Berkeley (1990) and conducted postdoctoral research at Northwestern I


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