A RuthenoceneâPNA Bioconjugate â Synthesis, Characterization

Article pubs.acs.org/bc

A Ruthenocene−PNA Bioconjugate  Synthesis, Characterization, Cytotoxicity, and AAS-Detected Cellular Uptake Annika Gross,#,† Nina Hüsken,#,† Julia Schur,‡ Łukasz Raszeja,† Ingo Ott,‡ and Nils Metzler-Nolte*,† †

Inorganic Chemistry I, Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Universitätsstrasse 150, D-44801 Bochum, Germany ‡ Institute of Medicinal and Pharmaceutical Chemistry, Technical University Braunschweig, Beethovenstrasse 55, D-38106 Braunschweig, Germany S Supporting Information *

ABSTRACT: Labeling of peptide nucleic acids (PNA) with metallocene complexes is explored herein for the modulation of the analytical characteristics, as well as biological properties of PNA. The synthesis of the first ruthenocene−PNA conjugate with a dodecamer, mixed-sequence PNA is described, and its properties are compared to a ferrocene-labeled analogue as well as an acetylated, metal-free derivative. The synthetic characteristics, chemical stability, analytical and thermodynamic properties, and the interaction with cDNA were investigated. Furthermore, the cytotoxicity of the PNA conjugates is determined on HeLa, HepG2, and PT45 cell lines. Finally, the cellular uptake of the metal-containing PNAs was quantified by high-resolution continuum source atomic absorption spectrometry (HR-CS AAS). An unexpectedly high cellular uptake to final concentrations of 4.2 mM was observed upon incubation with 50 μM solutions of the ruthenocene−PNA conjugate. The ruthenocene label was shown to be an excellent label in all respects, which is also more stable than its ferrocene analogue. Because of its high stability, low toxicity, and the lack of a natural background of ruthenium, it is an ideal choice for bioanalytical purposes and possible medicinal and biological applications like, e.g., the development of gene-targeted drugs.

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sequences as trans membrane carriers.18,19 A disadvantage of these peptide conjugates for in vivo applications is the often observed immunogeneity and the intrinsic toxicity of some of these peptides.20 In recent years, conjugates of metals or metal complexes and PNA oligomers have attracted great attention and were recently reviewed for the first time.21−23 Several examples thereof were reported to modify the PNA properties to an increased cellular uptake. Our group found an improved cellular delivery of a PNA conjugate with a cobalt complex,24 whereas Krämer et al. reported an enhanced uptake of Zn 2+ -modified PNA oligomers.25,26 The qualitative and quantitative detection of metal complex-conjugated biomolecules within the cell is greatly facilitated by the unique properties of the metal complexes. Specific examples include metalloimmunoassays,27−29 atomic absorption spectrometry (AAS),24 IR, fluorescence or optical spectroscopy,30−34 as well as RAMANand IR-microscopy.35−38 Thereby, especially an AAS-based quantification of the PNA uptake promises advantages over established biochemical methods due to its high sensitivity and metal-specificity.39 The high sensitivity and metal-specificity of AAS bears the potential to overcome limitations inherent in other methods such as the measurement of luciferase activity which is sensitive toward cell parameters like the cell density or

INTRODUCTION Peptide nucleic acids (PNA) are non-natural analogues of DNA and RNA with respect to their structure and hybridization properties. In PNA, the negatively charged (deoxy)ribose phosphodiester backbone of DNA/RNA is replaced by a neutral, pseudopeptide backbone of repeating N-(2aminoethyl)glycine units.1,2 Due to the noncharged backbone, PNA reveals outstanding hybridization properties, such as a thermodynamically and kinetically enhanced hybridization with DNA/RNA, and a high selectivity and excellent discrimination of single-nucleotide polymorphism (SNP). These features make PNA exceptionally attractive for analytical applications like the molecular recognition of nucleic acids.3−7 Medicinal and biological applications such as in antigene/antisense therapy8,9 or in drug discovery10 additionally benefit from the high chemical stability and its resistance toward nucleases and proteases.11 The main drawback of PNA regarding medicinal and biological applications is its poor intrinsic uptake, since unmodified PNA does not spontaneously enter the cell by passive diffusion through the cell membrane.12 Several strategies for an enhanced cellular uptake have been reported like, e.g., microinjection, electroporation, or cell permeabilization.13 Noninvasive methods usually use modified PNA oligomers with lipophilic moieties, negatively charged DNA complements, Trojan-peptides, and so forth, as trans membrane carriers.13−17 An efficient uptake is observed for PNA oligomers labeled with cell-penetrating peptides (CPP) like penetratin, Tat-derived peptides, transportan, or poly-Arg © 2012 American Chemical Society

Received: December 29, 2011 Revised: May 28, 2012 Published: July 24, 2012 1764

dx.doi.org/10.1021/bc200692g | Bioconjugate Chem. 2012, 23, 1764−1774

Bioconjugate Chemistry

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viability,18 or the fluorescent-microscopic detection of fluorescent-labeled PNA, which is restricted due to the ability of fluorophores to change the distribution and uptake of the PNA conjugate as well as influencing the cell viability.40−42 Most frequently, organo-metal complex/PNA conjugates are prepared by solid-phase synthesis. Our group has presented different methods for the conjugation of metal complexes in the course of the solid-phase synthesis of PNA, using the peptide coupling of carboxylic acid derivatives of the metal-complexes or the Cu-catalyzed [2 + 3] cycloaddition between azides and terminal alkynes (click chemistry) as the key reaction.43,44 The click chemistry approach enabled the introduction of one or even several (different) azido or alkynyl ferrocene derivatives to azide or alkyne functionalities at various positions within the PNA strand. With this chemistry, a target-oriented tuning of the properties of PNA could be realized.45−47 Ferrocene conjugates of PNA oligomers and monomers have also been reported by Baldoli et al.48−50 and Gasser et al.51,52 The labeling of PNA monomers and oligomers with different metal complexes besides ferrocene has also been investigated by Maiorana et al., who presented PNA conjugates with organometallic complexes of chromium,53,54 tungsten,55 and rhenium, as well as the radioactive technetium isotope 99mTc.56,57 The aim of this work is to provide a new metal−organic label for PNA sequences, which obtains good cellular uptake properties and is analytically detectable in living cells. Furthermore, such a label should be synthetically easily accessible and chemically stable and should not negatively influence neither the DNA binding properties nor the cell viability. A label with such properties is of high importance for the design of, e.g., new anticancer therapeutics or diagnostics but can be furthermore highly valuable also for antigene/ antisense agents in cell biology. As a promising organo-metallic complex regarding the desired properties, the ruthenium complex ruthenocene was chosen, which is the higher homologue of the well-studied ferrocene. Previously, our group presented a tris-(bipyridine)ruthenium(II) PNA conjugate, which shows interesting characteristics since the ruthenium complex is stable, undergoes reversible one-electron oxidation, and exhibits characteristic UV/vis absorption.44 On the other hand, the bulkiness of this ruthenium complex might hinder the cellular uptake of the PNA conjugate, which could explain why related metal derivatives exhibited a moderate antiproliferative effect.58 Therefore, although ruthenium has favorable properties per se, the tris(bipyridyl) complexes are far from ideal candidates as PNA labels for intracellular detection. In this paper, we report the facile solid-phase synthesis of a ruthenocenyl−PNA bioconjugate (Rc: ruthenocenyl, CpRuC5H4) via the peptide coupling of ruthenocenecarboxylic acid to the N-terminus of PNA. The Rc−PNA conjugate was comprehensively characterized by analytical techniques and the influence of the ruthenocene label onto the thermal stability of PNA•DNA duplexes was investigated in UV/vis-melting experiments. For possible medicinal or cell biological applications, the cellular uptake and the cytotoxicity of the Rc−PNA conjugate was studied. The experiments were additionally carried out with the analogue Fc−PNA conjugate (Fc: ferrocenyl, CpFeC5H4), as well as with an acetylated, metal-free PNA analogue.

Belgium), Aldrich/Sigma/Fluka (Deisenhofen, Germany), E. Merck (Darmstadt, Germany), Novabiochem (Laufelfingen, Switzerland), and IRIS Biotech (Marktredwitz, Germany) and were used without further purification. The preloaded polystyrene resin was purchased from Rapp Polymers (Tü bingen, Germany). Only L-amino acids were used throughout the synthesis. Fmoc/Bhoc-protected PNA monomers were purchased from Link Technologies (Lanarkshire, Scotland) and ASM (Hannover, Germany). All solutions were freshly prepared before use. HPLC fractions of all products were frozen in liquid nitrogen and lyophilized using a Christ Alpha 1−4 LD plus freeze−dryer. All aqueous solutions were prepared from Millipore water and filtered with a 0.22 μm syringe filter before use. Mass Spectrometry. MALDI-TOF mass spectra were recorded on a Daltonics Autoflex instrument (Bruker, Karlsruhe, Germany) in linear mode with positive polarity using sinapic acid as the matrix (6 mg/mL in ACN/water (+ 0.1% TFA) = 2:1). ESI mass spectra were recorded on an Esquire 6000 instrument (Bruker, Karlsruhe, Germany) applying the following parameters: sputtering voltage 4 kV; nebulizer pressure 10−20 psi; drying gas 5−10 L/min, 300 °C; flow rate 240 μL/h. For all mass spectrometric measurements, the PNA samples were prepared as 1 mg/mL solutions in water (+ 0.1% TFA). HPLC Analysis and Purification. HPLC analysis and purification were performed on a customized Knauer Smartline System (Knauer, Berlin, Germany), equipped with Knauer Smartline pumps 1000 and a Knauer Smartline UV detector 2550, using Reprosil-Pur C-18 reverse-phase columns (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) for analytical (250 × 4.6 mm) and preparative (250 × 10 mm) runs. Analytical (flow rate: 1.0 mL min−1) and preparative (flow rate: 4.0 mL min−1) runs were performed with a linear gradient of A (95% Millipore water, 5% ACN, 0.1% TFA (v/v/v)) and B (5% Millipore water, 95% ACN, 0.1% TFA (v/v/v)). Analytical runs: t = 0 min: 0% B; t = 18 min: 100% B; t = 22 min: 100% B; t = 30 min: 0% B. Preparative runs: t = 0 min: 0% B; t = 36 min: 100% B; t = 38 min: 100% B; t = 45 min: 0% B. All samples were filtered before injection using a 0.22 μm syringe filter. Spectra were recorded at 260 nm, and ambient temperature and retention times (tR/[min]) were noted in each case. Solid-Phase PNA Synthesis. SPPS was performed manually in 5 mL polypropylene one-way syringes as reaction vessels, which were equipped with a frit at the bottom. They were filled with 50 mg of polystyrene resin beads TentaGel R PHB Cys(Trt)Fmoc (0.18 mmol/g). The resin was swollen in DMF before use for 1 h. All reactions were performed on a mechanical shaker with 400 rpm, soaking approximately 3−4 mL of freshly prepared solutions into the syringe. Fmoc/Bhoc protected PNA monomers (5 equiv) were preactivated in Eppendorf tubes before every coupling step for 2 min with HATU (4.5 equiv) in DMF, adding DIPEA (0.2 M, 10 equiv), and 2,6-lutidine (0.3 M, 10 equiv) (A(Bhoc)-PNA-monomer: 5 min, C(Bhoc)-PNA-monomer: 7 min). The amino acids FmocAhx and Lys(Boc)Fmoc were preactivated in an analogous manner with TBTU and HOBt·H2O (4.5 equiv each). For each coupling step, the resin beads were treated with the activated acid under vibration (50 °C, 20 min) and subsequently washed with DMF. Every coupling step was monitored by the Kaiser test. Twofold Fmoc deprotection was performed with piperidine (20%, v/v) in DMF (2 min + 10 min). The resin

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EXPERIMENTAL SECTION General Experimental Conditions. All reagents and HPLC-grade solvents were purchased from Acros (Geel, 1765



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in an Eppendorf tube with HATU and HOBt·H2O (4.5 equiv each) in DMF, with DIPEA (0.2 M, 10 equiv) and 2,6-lutidine (0.3 M, 10 equiv). The resin was treated with the activated ferrocenecarboxylic acid under vibration (3 h, r.t.). The resin was then successively washed with DMF, DCM, and MeOH and subsequently shrunk by treating the resin with MeOH under vibration (10 min, r.t.). After draining off the solvent and removal of solvent residues under reduced pressure, the cleavage from the resin following the general procedure yielded FcPNA as a yellowish solid. FcPNA (M = 3784.5 g/mol): HPLC: tR = 10.26 min, 100%. MALDI-TOF MS: m/z (%) = 3785.5 [M+H]+ (100). ESI MS: m/z (%) = 1262.7 [M+3H]3+ (35.2), 947.4 [M+4H]4+ (100), 758.2 [M+5H]5+ (17.0), 632.0 [M+6H]6+ (2.9). UV/vis Melting Studies. Instrumentation. UV/vis measurements were performed with the UV/vis spectrophotometer Varian Cary 100 Conc (Varian Inc., Palo Alto, CA, USA), equipped with a 6 × 6 multicell block and Peltier thermostat and running with the Cary Win UV/vis software with Cary Bio package. Quartz Suprasil QS cuvettes (4 × 10 mm; Hellma, Müllheim, Germany) were used and densely sealed with Teflon caps for all measurements to avoid solvent evaporation at higher temperatures. The multicell block was purged with nitrogen gas during all measurements at lower temperatures (T < 15 °C). Determination of DNA and PNA Conjugate Concentrations. The UV/vis spectroscopic determination of the PNA conjugate concentration of the prepared stock solution (in 0.1 M PBS, pH 7.4) was performed from a diluted aliquot (1:10 dilution). The UV/vis spectrum (λ = 190−900 nm) was measured at 85 °C (after equilibration for 5 min to avoid PNA aggregation) versus a background of 0.1 M PBS (pH 7.4). The absorption A was read out at a wavelength of λ = 260 nm, to calculate the concentration c of the PNA solution according to the Beer−Lambert law A = ε l c (ε: molar extinction coefficient; l: cuvette path length; c: concentration of the PNA conjugate). The determination of the exact DNA concentration of the purchased DNA sequence was performed at T = 25 °C, otherwise in an analogous manner to the determination of the PNA concentration. Determination of the Molar Extinction Coefficient ε. The molar extinction coefficients ε of the PNA conjugates and DNA oligomers at λ = 260 nm were calculated from the incremental molar extinction coefficients εx of the nucleobases according to ε = ∑xnxεx (x = A/a, G/g, T/t, C/c, Fc or Rc; nx = number of monomer x in the oligomer). As proposed by Nielsen et al.,59 the incremental molar absorption coefficient of fully unstacked DNA nucleosides (at T = 25 °C)60 were taken as the incremental molar absorption coefficient of fully unstacked PNA nucleobases (requiring measurements at T > 80 °C), as reported in the literature (εa = 13 700 M−1 cm−1, εt = 8600 M−1 cm−1, εg = 11 700 M−1 cm−1, εc = 6600 M−1 cm−1, εFc = 9500 M−1 cm−1).60,61 The incremental molar absorption coefficient of the ruthenocene moiety εRc (λ = 260 nm, 0.1 M PBS, pH 7.4, T = 25 °C) was estimated using ruthenocenecarboxylic acid RcCO2H as a model compound. UV/vis spectra (λ = 190−900 nm) of a concentration series in the range of c = 10−500 μM with increments of c = 50−100 μM were recorded. The absorption was read out at λ = 260 nm and plotted against the respective concentration of ruthenocenecarboxylic acid, to give a straight line with R2 > 0.99995 (see Supporting Information). εRc was calculated from the slope of these straight lines according to the Beer−Lambert law, to result in εRc = 1310

beads were then washed successively with DMF, DCM, and DMF. The whole procedure (deprotection, coupling, monitoring) was repeated for every PNA monomer until the PNA sequence was completed. The synthesis of AcPNA, FcPNA and RcPNA, as described below, follows the Fmoc-deprotection of the last monomer of the completed PNA sequence. Final cleavage of the acetylated, ferrocenylated or ruthenocenylated PNA oligomer from the resin and the deprotection of all Bhoc, Boc, and Trt side chain protecting groups were simultaneously performed in TFA/TIS/phenol (85/5/10, v/v/v) (2 h + 1 h). Following the removal of TFA under reduced pressure, the crude product was precipitated with cold diethyl ether (−20 °C), washed twice with cold diethyl ether, and centrifuged each time (10 min, 8000 rpm). The obtained crude oligomer was lyophilized in acetonitrile/water, purified and analyzed with RPHPLC, and finally characterized with MALDI-TOF and ESI mass spectrometry. N-Terminally Acetylated PNA Oligomer (AcPNA). AcPNA was synthesized on 50 mg (10 μmol) of the resin TentaGel R PHB, which was preloaded with the 12-mer PNA sequence H-t c t a c g a g a c t c Lys Ahx CysOH. Therefore, an N-terminal double acetylation was performed subsequent to the Fmoc deprotection of the last PNA monomer of this sequence by treating the resin with a solution of acetic hydride (5%, v/v) and DIPEA (6%, v/v) in DMF for 2 × 3 min under vibration. The resin was then successively washed with DMF, DCM, and MeOH and subsequently shrunk by treating the resin with MeOH under vibration (10 min, r.t.). After draining off the solvent and removal of solvent residues under reduced pressure, the cleavage from the resin following the general procedure yielded AcPNA as a white solid. AcPNA (M = 3614.5 g/mol): HPLC: tR = 8.67 min, 100%. MALDI-TOF MS: m/z (%) = 3617.3 [M+H]+ (100). ESI MS: m/z (%) = 1206.0 [M+3H]3+ (25.5), 904.9 [M+4H]4+ (100), 724.2 [M+5H]5+ (17.3), 603.6 [M+6H]6+ (4.1). Ruthenocenoyl PNA Conjugate (RcPNA). The conjugation of ruthenocene via peptide coupling of ruthenocenecarboxylic acid was performed on 50 mg (10 μmol) of the resin TentaGel R PHB, which was preloaded with the 12-mer PNA sequence H-t c t a c g a g a c t c Lys Ahx CysOH, and follows the Fmoc deprotection of the last PNA monomer of this sequence. Ruthenocenecarboxylic acid (4 equiv) was preactivated for 3 min in an Eppendorf tube with HATU and HOBt·H2O (4 equiv each) in NMP, containing DIPEA (0.2 M, 6 equiv). The resin was treated with the activated ruthenocenecarboxylic acid under vibration (3 h, r.t.). The resin was then successively washed with DMF, DCM, and MeOH and subsequently shrunk by treating the resin with MeOH under vibration (10 min, r.t.). After draining off the solvent and removal of solvent residues under reduced pressure, the cleavage from the resin following the general procedure yielded RcPNA as an off-white solid. RcPNA (M = 3830.4 g/ mol): HPLC: tR = 10.27 min, 100%. MALDI-TOF MS: m/z (%) = 3831.9 [M+H]+ (100). ESI MS: m/z (%) = 1278.2 [M +3H]3+ (74.9), 958.7 [M+4H]4+ (100), 767.4 [M+5H]5+ (24.5). Ferrocenoyl PNA Conjugate (FcPNA). The conjugation of ferrocene via the peptide coupling of ferrocenecarboxylic acid was performed on 50 mg (10 μmol) of the resin TentaGel R PHB, which was preloaded with the 12-mer PNA sequence H-t c t a c g a g a c t c Lys Ahx CysOH, and follows the Fmoc deprotection of the last PNA monomer of this sequence. Ferrocenecarboxylic acid (5 equiv) was preactivated for 3 min 1766



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(±181) M−1 cm−1 from three independent measurements. Based on this, the molar extinction coefficients were calculated to ε(RcPNA) = 118 010 M−1 cm−1, ε(FcPNA) = 126 200 M−1 cm−1, ε(AcPNA) = 116 700 M−1 cm−1. The DNA concentration was calculated based on the following incremental molar absorption coefficients: εA = 15 300 M−1 cm−1, εT = 8700 M−1 cm−1, εG = 12 200 M−1 cm−1, εC = 7600 M−1 cm−1.62 UV/vis Melting Curves. For the measurement of PNA/DNA UV/vis melting curves, phosphate buffer solutions (0.1 M PBS, pH 7.4) of equimolar amounts of the single-stranded PNA conjugate RcPNA, FcPNA, or AcPNA and the single-stranded fully cDNA sequence G A G T C T C G T A G A with the final duplex concentration of c = 1.7 μM were prepared and filled into the quartz cuvettes. Multi-zeroing of the spectrophotometer was performed against cuvettes filled with phosphate buffer solution (0.1 M PBS, pH 7.4) at 25 °C. The controlled annealing of the PNA•DNA duplexes was performed by exposing the prepared PNA/DNA solutions to 85 °C for 5 min in the spectrophotometer and subsequently decreasing the temperature with a controlled cooling rate of 0.5 °C/min to a final temperature of 4 °C. Subsequently, the UV/vis melting curve was recorded at a wavelength of λ = 260 nm upon application of a temperature gradient ranging from T0 = 4 °C to T2.7 h = 85 °C with a heating rate of 0.5 °C/min. The collected raw data were saved as ASCII files and analyzed using the software ORIGIN. The obtained sigmoid-shaped melting curves were subjected to a polynomial data fit (9th order, 1000 data points) and subsequently differentiated (1st order). The melting point TM is equal to the maximum of the first derivative. Cell Culture. General Procedure. The human HepG2 cell line was obtained from “Deutsche Sammlung von Mikroorganismen und Zellkultur ACC 180”. The cell lines HeLa and PT45 were provided by Prof. Hahn (Molekulare Onkologie, Ruhr-University Bochum, Bochum, Germany). Cells were grown in RPMI 1640 with 1% sodium pyruvate, 1% Lglutamine, 100 units/mL Pen Strep, and 10% fetal bovine serum. The cells were maintained at 37 °C in a humidified incubator under an atmosphere containing 5% CO2. Wells used for HeLa cells were coated with 0.2% gelatin solution before use. HT-29 colon carcinoma cells were maintained in DMEM high glucose (PAA) supplemented with 50 mg/L gentamycin and 10% (v/v) fetal calf serum (FCS) prior to use. Cytotoxicity Experiments. To determine the activity of RcPNA and FcPNA in comparison to AcPNA, two antiproliferative assays, resazurin and crystal violet, were performed. 6000 cells/well of HeLa, PT45, and HepG2 cells were incubated in 96-well plates at 37 °C and 5% CO2 for 24 h. To determine the initial cell viability and biomass, a t0-plate with HeLa, HepG2, and PT45 was additionally plated, and after 24 h incubation, the resazurin assay was performed. Subsequently, the cells were fixed with 0.2% glutardialdehyde solution, followed by the crystal violet assay. In the remaining plates the medium was replaced with medium containing AcPNA, FcPNA, or RcPNA with 0.5% DMSO added to improve the solubility of the compound. Concentrations between 1 and 200 μM were used and each concentration was tested six times in parallel. As a positive control, cisplatin was used, and as negative control, medium containing 0.5% DMSO. To determine the effect of metallocene carboxylic acid, ferrocene- and ruthenocenecarboxylic acid were tested up to a concentration of 1 mM under the same conditions as the metallocene−PNA conjugates. The incubation time of the

compounds was 48 h. Then, the compound-containing medium was removed, the cells were washed with PBS, and the resazurin assay was performed. Resazurin Assay. 1 mL of resazurin solution per plate was diluted with 9 mL medium without phenol red. 100 μL of this solution was added per well and the plates were measured using a Tecan plate reader at 600 nm, using a reference wavelength of 690 nm. The plates were incubated for 2 h at 37 °C and 5% CO2 and were measured again. Crystal Violet Assay. To perform the crystal violet assay, the resazurin solution was removed and the cells were fixed using 0.2% glutardialdehyde for 25 min. The glutardialdehyde solution was removed and exchanged for 100 μL of 0.1% triton solution. After a short incubation, all liquids were removed and the fixed cells were stained with a 0.02 M crystal violet solution for 30 min. Afterward, the wells were washed intensely with H2O and were filled with 100 μL of 96% ethanol, followed by shaking on a softly rocking rotary shaker for 3 h. The absorption of the ethanolic solution was measured using a microplate reader at 570 nm. The mean absorption of the initial cell plate (t0-plate) for each cell line was subtracted from the absorption of each experiment and control. For both assays, the negative control was set to 100%. Cellular Uptake. For cellular uptake studies, HT-29 colon carcinoma cells were grown until at least 70% confluency in 75 cm2 cell culture flasks. Stock solutions of the metallocene−PNA conjugate, namely, FcPNA or RcPNA, in demineralized water were freshly prepared and diluted with cell culture medium to the desired concentration of 50 μM. The cell culture medium of the cell culture flasks was replaced with 10 mL of the cell culture medium solutions containing the metallocene−PNA conjugate and the flasks were incubated at 37 °C/5% CO2 for 6 h. Subsequent to the incubation period, the culture medium was removed, and the cell layer washed with 10 mL PBS, treated with 2 mL trypsin solution (0.05% trypsin in PBS), and incubated for 2 min at 37 °C/5% CO2 after removal of the trypsin solution. Cells were resuspended in 10 mL PBS and cell pellets were isolated by centrifugation (RT, 5400 rpm, 5 min). Cellular lysates were prepared by resuspending an isolated cell pellet in 1 mL demineralized water followed by ultrasonication. An aliquot was removed for the purpose of protein quantification by the method of Bradford.63 The metal content of the samples was determined by high-resolution continuum source atomic absorption spectrometry (HR-CS AAS, see below). Results were calculated from the data of two independent experiments and are expressed as pmol of the metallocene−PNA conjugate per milligram of cellular protein. On the basis of the knowledge of the cellular diameter, the values were also estimated as intracellular molar metal concentrations.64 HR-CS AAS Measurements. A contrAA 700 highresolution continuum source atomic absorption spectrometer (Analytik Jena AG) was used for the metal measurements. Iron was measured at a wavelength of 248.37 nm; ruthenium was quantified at a wavelength of 349.90 nm. Because of the high iron background level in biological material, matrix-matched calibration was used for iron quantification in the FcPNA samples. For this purpose, the protein levels of all samples and standards were adjusted to the same concentration by dilution with distilled water. For ruthenium measurements in the RcPNA samples, aqueous standards were used for calibration purpose. To 200 μL of all probes and standards each, 20 μL Triton X-100 (1%) and 20 μL nitric acid (13%) were added. 1767



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Scheme 1. Synthesis Scheme for the Solid-Phase Synthesis of Metallocene-PNA Conjugates FcPNA and RcPNAa

a (a) Fmoc deprotection: piperidine/DMF (20%, v/v); (b) Coupling Fmoc-AA-OH (5 equiv): TBTU, HOBt·H2O (4.5 equiv each), DIPEA, 2,6lutidine (10 equiv each) in DMF; (c) coupling Fmoc-PNA(Bhoc)-OH (5 equiv): HATU (4.5 equiv), DIPEA, 2,6-lutidine (10 equiv each) in DMF; (d) coupling Fc-CO2H (5 equiv): HATU, HOBt·H2O (4.5 equiv each), DIPEA, 2,6-lutidine (10 equiv each) in DMF; coupling Rc-CO2H (4 equiv): HATU, HOBt·H2O (4 equiv each), DIPEA (6 equiv) in NMP; (e) Cleavage: TFA/TIS/phenol (85/5/10, v/v/v). See Experimental Section for further details.

Samples were injected at a volume of 25 μL into coated standard graphite tubes (“AAS IC-Standardrohr, beschichtet”, Analytik Jena AG). The optimized temperature time program for iron in the graphite tube is given in the Supporting Information (Table S1). For ruthenium, a furnace program as described in the literature was used.65 During the temperature program, the graphite tube was purged with a constant argon gas flow, which was only halted during the zeroing and atomization steps. The mean integrated absorbances of triple injections were used throughout the study.

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RESULTS AND DISCUSSION Synthesis, Characterization, and Chemical Stability. The 12-mer PNA sequence of the PNA conjugates RcPNA, FcPNA, and AcPNA was synthesized according to Scheme 1 by manual standard Fmoc solid-phase synthesis on a FmocCys(Trt) preloaded TentalGel R PHB resin using Fmoc/Bhocprotected PNA monomers (see Experimental Section for details).45 The chosen PNA sequence is a mixed sequence of all four nucleobases and targets a specific region in the central domain of the 16S rRNA of E. coli. A peptidic linker is bound to the PNA C-terminus, which contains a lysine residue for increased solubility and enhanced cellular uptake of the PNA conjugate.16,45 The metallocene complex was conjugated to the PNA N-terminus, since an undesired decrease in PNA•DNA duplex stability was observed upon modification of internal strand positions of the PNA sequence.66,67 Ferrocene and ruthenocene were conjugated to the deprotected N-terminus of the PNA oligomer on the solid support by performing peptide coupling with ferrocene carboxylic acid (Fc-CO2H) and ruthenocene carboxylic acid (Rc-CO2H), respectively, as the sequence-terminating coupling step.45,68 The success of the coupling was monitored by the Kaiser test69 and later proven by HPLC of the crude compound (Figure 1). For improved solubility of Rc-CO2H, NMP was used instead of DMF. Cleavage from the resin and the removal of all protecting groups was performed with TFA/TIS/phenol 85:5:10 (v/v/v), and precipitation with diethyl ether followed by centrifugation

Figure 1. Analytical data of RcPNA. (A) MALDI-TOF spectrum; enlarged section shows the isotopic pattern of the base peak at m/z = 3831.9 (m/z-scale normalized to m/z of the base peak; continuous black line: measured pattern, dashed red line: isotope pattern simulated with the software Bruker Daltonics DataAnalysis 3.4 (Bruker, Karlsruhe, Germany). (B) HPLC chromatogram detected at a wavelength of λ = 260 nm.

gave the crude product of RcPNA as a white solid and of FcPNA as a yellowish solid. The N-terminally acetylated PNA oligomer AcPNA was synthesized as a metal-free analogue of FcPNA and RcPNA. 1768



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The crude products were purified by preparative HPLC (see Experimental Section). Analytical HPLC proved high purity of the three compounds and MALDI-TOF mass spectrometry showed only the respective [M+H]+ peak for all compounds. In ESI-MS, all PNA conjugates showed the respective [M+4H]4+ peak as the base peak with an excellent mass accuracy (deviation from theoretical value 98%. On the basis of HPLC and MS, it can be thus concluded that all solid-phase reaction steps during the synthesis of RcPNA, FcPNA, and AcPNA including the coupling of Rc-CO2H and Fc-CO2H occurred with nearly complete conversion. Magnification of the [M+H]+ base peak in the MALDI-TOF spectra of RcPNA furthermore revealed a well-resolved isotope distribution (see Figure 1A), which is in good agreement with the calculated isotope pattern of RcPNA and hence is a distinct indicator for the presence of ruthenocene. The analytical characterization of the crude compounds furthermore indicates that both metallocene labels are completely stable to the fairly harsh TFA cleavage conditions. However, the ferrocene label turned out to be somewhat unstable upon extended storage even at −20 °C after HPLC purification, as shown by the MS detection of a molecular fragment resulting from the loss of the FeCp group (data not shown). The formation of this fragment can be explained by primary oxidation and subsequent degradation of ferrocene induced by nucleophilic agents, which is a wellknown degradation pathway for ferrocene.70 An analogous degradation was never observed for RcPNA. This observation is in line with the higher oxidation potential of ruthenocene vs ferrocene and indicates an improved stability in comparison to FcPNA. Analytical HPLC of the three conjugates furthermore shows similar retention times for the metal−PNA conjugates of tR(RcPNA) = 10.27 min and tR(FcPNA) = 10.26 min, whereas AcPNA exhibits a smaller value of tR(AcPNA) = 8.67 min. Conjugation of ferrocene or ruthenocene to PNA oligomers thus induces an increase in the lipophilicity of the PNA oligomer. DNA Binding. UV/vis melting experiments were performed in order to study the impact of the different N-terminally attached metallocene labels onto the thermodynamic stability of the corresponding fully complementary PNA•DNA duplexes. Melting experiments were performed by running a temperature profile from T = 5−85 °C with detection at a wavelength of λ = 260 nm (see Figure 2). The determined TM values equal the maximum of the melting curves’ first derivative. The PNA•DNA duplexes of FcPNA and RcPNA exhibit values of TM(FcPNA•DNA) = 60.1 (±0.3) °C and TM(RcPNA•DNA) = 60.5 (±0.2) °C, which are about 2.5 and 2.1 °C, respectively, lower than the value of TM(AcPNA•DNA) = 62.6 (±0.3) °C determined for the acetylated analogue. The presence of the N-terminally bound metallocene complex hence slightly destabilizes the duplex structures. As a possible reason, an impediment of the nucleobase-pairing at the N-terminus under formation of a “frayed” strand-end is proposed, which has already been discussed for a N-terminally bound tris-(bipyridine)ruthenium(II) complex and is furthermore documented for terminal

Figure 2. Overlay of UV/vis-melting curves of RcPNA, FcPNA, and AcPNA (absorbance is documented as the percentaged fraction of the absorbance of the fully unstacked, single-stranded species at T = 80 °C).

single base pair mismatches.44,71 This thermodynamic destabilization is underscored by differences in the related hypochromic effect during duplex formation. Whereas the AcPNA•DNA duplex exhibited a hypochromic effect of 12.3% upon duplex formation (comparing T = 80 and 5 °C), the metallocene PNA conjugates FcPNA•DNA and RcPNA•DNA show smaller values of 10.8% and 10.5%, respectively. Since the hypochromic effect is related to the extent of π−π nucleobase stacking interactions, a smaller hypochromic effect supports the notion of metallocene-induced attenuation of the π−π stacking.44 Antiproliferative Effect and Cellular Uptake Studies. The biological properties of the metallocene−PNA conjugates were examined as a basis for potential future applications as therapeutic or diagnostic agents, for instance, in cancer therapy. First, the cellular uptake of the metallocene−PNA conjugates was studied. To this end, the cellular metal concentrations were quantified by high-resolution continuum source atomic absorption spectrometry (HR-CS AAS), which is a novel AAS technology offering a simultaneous background correction besides a high specificity and low detection limits for metals.39 Additionally, the cytotoxicity of all PNA conjugates was studied in order to determine a possible damage of the cell membrane due to apoptotic or necrotic effects, which would lead to artificially increased values for cellular uptake. Cytotoxicity. The antiproliferative effects of RcPNA and FcPNA were studied in comparison to AcPNA using the resazurin assay and additionally the crystal violet assay. These two assays evaluate different cell parameters. While the resazurin assay determines the cell viability, the crystal violet assay gives information about the cell biomass. The commonly used human liver carcinoma cell line HepG2 and the cell line PT45, a pancreatic cancer cell line of high interest due to the profound resistance to treatment of pancreatic cancer, were chosen for comparative cytotoxicity measurements of all PNA conjugates. RcPNA was additionally tested on HeLa cells, a cervical cancer cell line which in general reveals the highest sensitivity of the studied cell lines. Both assays showed that none of the compounds had any cytotoxic effect after an incubation time of 48 h and up to a concentration of 200 μM (see Figure 3). The absence of an antiproliferative effect of the metallocenelabeled conjugates FcPNA and RcPNA furthermore indicates that the metallocene groups do not increase the cytotoxicity of the PNA. This finding is supported by the absence of any 1769



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Figure 3. Proliferation of PT45, HepG2, and HeLa cells in the presence of varying concentrations of RcPNA (in medium with 0.5% DMSO), as determined by the crystal violet assay. Figure 4. HR-CS AAS signal induced by FcPNA and RcPNA, respectively. The iron or ruthenium absorption signal can be measured at 248.37 nm (iron, FcPNA) or 349.90 nm (ruthenium, RcPNA) without background interferences. RcPNA shows a broad signal caused by its low volatility. In both cases, quantification was possible using the absorbances (correlation coefficient of standard curve >0.995).

cytotoxic activity of the free compounds ferrocene- and ruthenocenecarboxylic acid up to a concentration of 1 mM. While several ferrocene-containing compounds with strong antiproliferative activity are reported in the literature,72,73 the cytotoxicity of compounds conjugated with a simple ferrocene amide moiety, as is present in FcPNA, is usually fairly low. The low toxicity of RcPNA is readily rationalized since ruthenocene is chemically and oxidative more stable than ferrocene,74 limiting its possible interactions with biological material. This outcome is supported by our previous findings, which did not show any cytotoxic effect for ruthenocenecarboxylic acid or ruthenocene containing peptides.75 Recently, Williams et al. reported ruthenocene analogues with a moderate to weak cytotoxicity. However, the cytotoxic effect of these derivatives appeared to be governed by the substituents of the ruthenocene rather than the metal complex itself.76 Cellular Uptake. The cellular uptake of the metallocene− PNA conjugates FcPNA and RcPNA was studied by incubating HT-29 colon carcinoma cells. The incubation was performed with 50 μM solutions of the respective conjugate in cell culture medium over a period of 6 h. The uptake of the metal compounds into the cells was quantified by determination of the iron and ruthenium content, respectively, by HR-CS AAS (see Figure 4). The cellular protein (CP) content of the same samples was measured simultaneously according to the method of Bradford.63 Finally, the determined metal content was correlated to the cell protein content (pmol metal per μg CP). The analysis revealed surprisingly high cellular uptake levels for the metallocene−PNA conjugates with a cellular iron concentration of 72 pmol/μg CP for FcPNA and a cellular ruthenium concentration of 21 pmol/μg CP for RcPNA. The determination of the cellular concentration of FcPNA, despite the high natural background of iron, was possible due to this exceptionally strong uptake of the iron-containing complex. On the basis of these values, the intracellular concentrations of FcPNA and RcPNA can be estimated on the basis of distinct cellular parameters (e.g., mean content of cellular protein, mean cell volume).64 Both compounds revealed relatively high intracellular concentrations in the low millimolar range (14.2 mM for FcPNA and 4.2 mM for RcPNA, see Table 1). With this, the intracellular concentration of both PNA conjugates significantly exceeds the extracellular concentration of 50 μM, corresponding to a 284-fold accumulation for FcPNA compared to extracellular cell culture medium and an 83-fold

cellular accumulation for RcPNA. This may indicate that the cellular uptake occurs according to an active uptake process rather than an unspecific diffusional process. This is an important finding regarding the fact that unmodified PNA is assumed to generally reveal a poor intrinsic uptake, and furthermore, no peptide modification according to classical noninvasive delivery strategies had been performed for the preparation of the compounds tested here. However, an increased intracellular concentration of a comparable octamer PNA sequence labeled with a cobalt complex ([Co(bpa)]-CH2(p-C6H4)-C(O)-Ahx-tgttatcc-Lys-NH2) had been already reported by our group.24 For this compound, a cellular uptake of ∼0.18 pmol/μg CP with an intracellular concentrations of >70 μM was measured in comparison to the extracellular concentration of 50 μM (cell line: HT-29 colon carcinoma cells; incubation time: 6 h). The cellular uptake of the compounds presented here seems to be significantly improved to reach for RcPNA a 117-fold and for FcPNA a 400-fold accumulation if compared to the cobalt complex. Comparable high uptake values of 150-fold accumulation in the cell were presented by Ott et al. for Co-ASS (a cobalt carbonyl containing acetylsalicylic acid derivative) after 6 h incubation in MCF-7 cells.77 An important matter of discussion is the reason for the high cellular uptake of FcPNA and RcPNA. In comparison to previously reported PNA oligomers, a relatively good cellular uptake may be expected due to the short sequence length of just twelve oligonucleotides and most notably the positively charged lysine moiety, which was introduced into the peptide residue as an uptake enhancer. Moreover, the lipophilic metallocenes also seem to have a decisive effect on the cellular accumulation. Comparison between the cellular uptake values of FcPNA and RcPNA reveals a 3.4-fold larger value for FcPNA. While the overall structures of the two metallocenes are rather similar, ruthenocene has a larger volume than ferrocene (mean values for Fe−C distance: 2.048 Å, Ru−C distance: 2.208 Å).78,79 Under the assumption of a more 1770



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Table 1. Experimental Data of AcPNA, FcPNA, and RcPNA conjugate

TM/°Ca

ΔAbs/%a

tR/minb

metal/protein/μg mg−1c

cell concentration/mMc

AcPNA FcPNA RcPNA

62.6 (±0.3) 60.1 (±0.3) 60.5 (±0.2)

12.3 (±0.1) 10.8 (±0.1) 10.5 (±0.2)

8.67 10.26 10.27

− 4.05 ± 0.26 2.15 ± 0.10

− 14.21 ± 0.91 4.16 ± 0.18

Analysis of the UV/vis melting experiments of the fully complementary PNA•DNA duplexes of the conjugates at λ = 260 nm. bDetermined by HPLC. cCellular uptake into HT-29 cells after 6 h incubation time determined by HR-CS AAS.

a

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intricate and active uptake mechanism, a further impact could be attributed to the well-known differences in the chemical as well as electrochemical reactivity of ferrocene and ruthenocene.80 In contrast, the impact of differences on the lipophilicity can be excluded based on the nearly identical HPLC retention times of tR(RcPNA) = 10.27 min and tR(FcPNA) = 10.26 min (see Table 1).

ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatograms, ESI- or MALDI-TOF spectra, and cytotoxicity assays (crystal violet and resazurin) for all PNA conjugates, UV-analysis for the determination of the extinction coefficient of Rc-CO2H, UV-melting curves of the fully cDNA duplexes of all PNA conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSIONS The first ruthenocene−PNA bioconjugate was synthesized and analyzed in this work in order to evaluate the suitability of ruthenocene−PNA conjugates for bioanalytical or medicinal applications like, e.g., as gene-targeted drugs or in antigene/ antisense therapy. A comparative study with its ferrocene derivative, as well as nonmetallic acetylated analogue furnished the following results: (1) The solid-phase synthesis of RcPNA proved to be fully comparable to the synthesis of FcPNA and AcPNA regarding conversion, purity and yield. (2) The thermodynamic stability of RcPNA is comparable to that of the metal-free analogue AcPNA and superior to that of FcPNA, which can be explained by the different redox properties of the metal core. (3) The thermal stability of the DNA duplex of RcPNA is comparable to that of the FcPNA analogue, and only slightly lower than that of the corresponding AcPNA•DNA duplex. This effect is ascribed to a steric hindrance by the metallocene complex. The biological activity of the PNA conjugates was examined by studying their cytotoxicity and cellular uptake in different cell lines. All PNA conjugates were found to be nontoxic, indicating that the metallocene headgroup does not enhance the cytotoxicity of the nontoxic PNA sequence. Consequently, no cell damage distorts the results from the cellular uptake study. HR-CS AAS-quantification of the cellular uptake of FcPNA and RcPNA revealed cellular concentrations of both conjugates which are surprisingly high compared to literature values. Hence, a significant impact on the high uptake values can be ascribed to the presence of the metallocene group, with only minor differences between the two metal labels. However, further sequence characteristics have to be considered before a general pattern for the cellular uptake characteristics of both labels in bioconjugates can be derived. Bioanalytical benefits of the ruthenocene−PNA conjugate in comparison to its ferrocene-labeled analogue are related to the absence of any natural background for ruthenium. A low natural background is especially important when using standard AASinstead of HR-CS technology. Furthermore, potential detection and quantification by mass spectrometry is possible based on the metal’s specific isotope pattern in all Ru-containing bioconjugates. All taken together, the favorable properties of ruthenocene-containing PNA bioconjugates as demonstrated herein make this class of compounds especially attractive for medicinal and biological applications.

AUTHOR INFORMATION

Corresponding Author

*Fax: +49 (0)234 32 14378; Tel: +49 (0)234 32 28152; Email: [email protected]. Author Contributions #

These authors have decided to publish this paper with shared first authorship and listing in alphabetical order. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.G. and N.H. thank the Ruhr-University Research School funded by Germany’s Excellence Initiative [DFG GSC 98/1] for financial support. The authors thank Prof. Stephan Hahn and his co-workers for providing the HeLa, PT45, and HepG2 cells and cell laboratory facilities. Financial support from the German Science Foundation DFG (FOR 630) is also gratefully acknowledged.

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ABBREVIATIONS A, absorption; AAS, atomic absorption spectrometry; ACN, acetonitrile; Ahx, amino hexanoic acid; ASS, acetylsalicylic acid; bpa, 1,2-di(4-pyridyl)ethane; Bhoc, benzhydryl-oxycarbonyl; Boc, tert-butyl-oxycarbonyl; c, concentration; cp, cyclopentadiene; CP, cellular protein; DCM, dichloromethane; DIPEA, diisopropyl-ethylamine; DMF, N,N-dimethyl formamide; DMSO, dimethyl sulfoxide; ε, molar extinction coefficient; ESI, electrospray ionization; Fc, ferrocenyl (C5H5FeC5H4); FcH, ferrocene (dicyclopentadienyl iron, Cp2Fe, (C5H5)2Fe); Fmoc, fluorenylmethoxy-carbonyl; HATU, 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxy-1H-benzotriazole; HPLC, high performance liquid chromatography; HR-CS, high-resolution continuum source; l, cuvette path length; MALDI-TOF, matrix assisted laser desorption/ionization - time-of-flight; MeOH, methanol; NMP, N-methyl-2-pyrrolidon; PBS, phosphate buffered saline; PHB, photochemical hole burning; Rc, ruthenocenyl (C5H5RuC5H4); RcH, ruthenocene; RP, reversed phase; rRNA, ribosomal RNA; PDA, photodiode array; PNA, peptide nucleic acids; SNP, single-nucleotide polymorphism; SPPS, solid phase peptide (or PNA) synthesis; TBTU, 2-(1Hbenzotriazole-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid; TIS, triisopropyl silane; TM, melting temperature value; tR, retention time; The common three-letter 1771



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uptake of PNA, peptides, and PNA-peptide conjugates. Antisense Nucleic Acid Drug Dev. 12, 51−63. (20) Jones, S. W., Christison, R., Bundell, K., Voyce, C. J., Brockbank, S. M. V., Newham, P., and Lindsay, M. A. (2005) Characterisation of cell-penetrating peptide-mediated peptide delivery. Br. J. Pharmacol. 145, 1093−1102. (21) Gasser, G., Sosniak, A. M., and Metzler-Nolte, N. (2011) Metalcontaining peptide nucleic acid conjugates. Dalton Trans. 40, 7061− 7076. (22) van Staveren, D. R., and Metzler-Nolte, N. (2004) Bioorganometallic chemistry of ferrocene. Chem. Rev. 104, 5931−5985. (23) Salmain, M., and Metzler-Nolte, N. (2008) The bioorganometallic chemistry of ferrocene, in Ferrocenes (Stepnicka, P., Ed.) pp 499− 639, John Wiley & Sons, Chichester, UK. (24) Kirin, S. I., Ott, I., Gust, R., Mier, W., Weyhermueller, T., and Metzler-Nolte, N. (2008) Cellular uptake quantification of metalated peptide and peptide nucleic acid bioconjugates by atomic absorption spectroscopy. Angew. Chem., Int. Ed. 47, 955−959. (25) Fuessl, A., Schleifenbaum, A., Goeritz, M., Riddell, A., Schultz, C., and Kraemer, R. (2006) Cellular uptake of PNA-terpyridine conjugates and its enhancement by Zn2+ ions. J. Am. Chem. Soc. 128, 5986−5987. (26) Metzler-Nolte, N. (2006) Conjugates of peptides and PNA with organometallic complexes: syntheses and applications, in Bioorganometallics (Jaouen, G., Ed.) pp 125−179, Wiley-VCh, Weinheim, Germany. (27) Cais, M., Slovin, E., and Snarsky, L. (1978) Metalloimmunoassay II. Iron-metallohaptens from estrogen steroids. J. Organomet. Chem. 160, 223−230. (28) Cais, M., Dani, S., Eden, Y., Gandolfi, O., Horn, M., Isaacs, E. E., Josephy, Y., Saar, Y., Slovin, E., and Snarsky, L. (1977) Metalloimmunoassay. Nature 270, 535−536. (29) Forrow, N. J., Foulds, N. C., Frew, J. E., and Law, J. T. (2004) Synthesis, characterization, and evaluation of ferrocene-theophylline conjugates for use in electrochemical enzyme immunoassay. Bioconjugate Chem. 15, 137−144. (30) Vessières, A., Salmain, M., Brossier, P., and Jaouen, G. (1999) Carbonylmetallo immuno assay: a new application for Fourier transform infrared spectroscopy. J. Pharm. Biomed. Anal. 21, 625−633. (31) Salmain, M., Vessières, A., Brossier, P., Butler, I. S., and Jaouen, G. (1992) Carbonylmetalloimmunoassay (CMIA) a new type of nonradioisotopic immunoassay. J. Immunol. Methods 148, 65−75. (32) Guillena, G., Halkes, K. M., Rodriguez, G., Batema, G. D., van Koten, G., and Kamerling, J. P. (2003) Organoplatinum(II) complexes as a color biomarker in solid-phase peptide chemistry and screening. Org. Lett. 5, 2021−2024. (33) Guillena, G., Rodriguez, G., Albrecht, M., and van Koten, G. (2002) Covalently bonded platinum(II) complexes of alpha-amino acids and peptides as a potential tool for protein labeling. Chem. Eur. J. 8, 5368−5376. (34) Raszeja, L., Maghnouj, A., Hahn, S., and Metzler-Nolte, N. (2011) A novel organometallic Re(I) complex with favourable properties for bioimaging and applicability in solid-phase peptide synthesis. ChemBioChem 12, 371−376. (35) Meister, K., Niesel, J., Schatzschneider, U., Metzler-Nolte, N., Schmidt, D. A., and Havenith, M. (2010) Label-free imaging of metalcarbonyl complexes in live cells by raman microspectroscopy. Angew. Chem., Int. Ed. 49, 3310−3312. (36) Kong, K. V., Chew, W., Lim, L. H. K., Fan, W. Y., and Leong, W. K. (2007) Bioimaging in the mid-infrared using an organometallic carbonyl tag. Bioconjugate Chem. 18, 1370−1374. (37) Yan, S. X., Seidel, M. T., Zhang, Z. Y., Leong, W. K., and Tan, H. S. (2011) Ultrafast vibrational relaxation dynamics of carbonyl stretching modes in Os3(CO)12. J. Chem. Phys. 135, 024501/1− 024501/10. (38) Li, C., and Leong, W. K. (2008) The deposition of osmium carbonyl clusters onto inorganic oxide surfaces: A ToF-SIMS and IR spectroscopic study of the surface species. J. Colloid Interface Sci. 328, 29−33.

code for amino acids is used throughout. Only enantiomerically pure L amino acids were used. Nucleobases are denoted by their usual abbreviations in DNA; small letters, however, indicate PNA monomers

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REFERENCES

(1) Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497−1500. (2) Egholm, M., Nielsen, P. E., Buchardt, O., and Berg, R. H. (1992) Recognition of guanine and adenine in DNA by cytosine and thymine containing peptide nucleic acids (PNA). J. Am. Chem. Soc. 114, 9677− 9678. (3) Wang, J., Paleček, E., Nielsen, P. E., Rivas, G., Cai, X., Shiraishi, H., Dontha, N., Luo, D., and Farias, P. A. M. (1996) Peptide nucleic acid probes for sequence-specific DNA biosensors. J. Am. Chem. Soc. 118, 7667−7670. (4) Hüsken, N., Gębala, M., La Mantia, F., Schuhmann, W., and Metzler-Nolte, N. (2011) Mechanistic studies of Fc-PNA(•DNA) surface dynamics based on the kinetics of electron-transfer processes. Chem. Eur. J. 17, 9678−9690. (5) Hüsken, N., Gębala, M., Battistel, A., La Mantia, F., Schuhmann, W., and Metzler-Nolte, N. (2011) Impact of single basepair mismatches on electron-transfer processes at Fc-PNA•DNA modified gold surfaces. ChemPhysChem 13, 131−139. (6) Hüsken, N., Gębala, M., Schuhmann, W., and Metzler-Nolte, N. (2010) A single-electrode, dual-potential ferrocene−PNA biosensor for the detection of DNA. ChemBioChem 11, 1754−1761. (7) Maurer, A., Kraatz, H.-B., and Metzler-Nolte, N. (2005) Synthesis and electrochemical characterization of metallocene-PNA oligomers. Eur. J. Inorg. Chem., 3207−3210. (8) Wang, G., and Xu, X. S. (2004) Peptide nucleic acid (PNA) binding-mediated gene regulation. Cell Res. 14, 111−116. (9) Knudsen, H., and Nielsen, P. E. (1996) Antisense properties of duplex- and triplex-forming PNAs. Nucleic Acids Res. 24, 494−500. (10) Good, L., and Nielsen, P. E. (1997) Progress in developing PNA as a gene-targeted drug. Antisense Nucleic Acid Drug Dev. 7, 431−437. (11) Demidov, V. V., Potaman, V. N., Frank-Kamenetskii, M. D., Egholm, M., Buchardt, O., Sönnichsen, S. H., and Nielsen, P. E. (1994) Stability of peptide nucleic acids in human serum and cellular extracts. Biochem. Pharmacol. 48, 1310−1313. (12) Wittung, P., Kajanus, J., Edwards, K., Nielsen, P., Norden, B., and Malström, B. G. (1995) Phospholipid membrane permeability of peptide nucleic acid. FEBS Lett. 365, 27−29. (13) Koppelhus, U., and Nielsen, P. E. (2003) Cellular delivery of peptide nucleic acid (PNA). Adv. Drug Delivery Rev. 55, 267−280. (14) Ljungstrøm, T., Knudsen, H., and Nielsen, P. E. (1999) Cellular uptake of adamantyl conjugated peptide nucleic acids. Bioconjugate Chem. 10, 965−972. (15) Chinnery, P. F., Taylor, R. W., Diekert, K., Lill, R., Turnbull, D. M., and Lightowlers, R. N. (1999) Peptide nucleic acid delivery to human mitochondria. Gene Ther. 6, 1919−1928. (16) Albertshofer, K., Siwkowski, A. M., Wancewicz, E. V., Esau, C. C., Watanabe, T., Nishihara, K. C., Kinberger, G. A., Malik, L., Eldrup, A. B., Manoharan, M., Geary, R. S., Monia, B. P., Swayze, E. E., Griffey, R. H., Bennett, C. F., and Maier, M. A. (2005) Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid. J. Med. Chem. 48, 6741− 6749. (17) Marin, V. L., Roy, S., and Armitage, B. A. (2004) Recent advances in the development of peptide nucleic acid as a gene-targeted drug. Expert Opin. Biol. Ther. 4, 337−348. (18) Bendifallah, N., Rasmussen, F. W., Zachar, V., Ebbesen, P., Nielsen, P. E., and Koppelhus, U. (2006) Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA). Bioconjugate Chem. 17, 750−758. (19) Koppelhus, U., Awasthi, S. K., Zachar, V., Holst, H. U., Ebbesen, P., and Nielsen, P. E. (2002) Cell-dependent differential cellular 1772



Article

(39) Welz, B., Becker-Ross, H., Florek, S., and Heitmann, U. (2005) High-resolution continuum source AAS, Wiley-VCH, Weinheim, Germany. (40) Lundin, P., Johansson, H., Guterstam, P., Holm, T., Hansen, M., Langel, U., and El Andaloussi, S. (2008) Distinct uptake routes of cellpenetrating peptide conjugates. Bioconjugate Chem. 19, 2535−2542. (41) Fischer, R., Waizenegger, T., Kohler, K., and Brock, R. (2002) A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import. Biochim. Biophys. Acta, Biomembr. 1564, 365−374. (42) El-Andaloussi, S., Jaerver, P., Johansson, H. J., and Langel, U. (2007) Cargo-dependent cytotoxicity and delivery efficacy of cellpenetrating peptides: A comparative study. Biochem. J. 407, 285−292. (43) Hess, A., and Metzler-Nolte, N. (1999) Transition metal labels on peptide nucleic acid (PNA) monomers. Chem. Commun., 885−886. (44) Verheijen, J. C., van der Marel, G. A., van Boom, J. H., and Metzler-Nolte, N. (2000) Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding. Bioconjugate Chem. 11, 741−743. (45) Hüsken, N., Gasser, G., Köster, S. D., and Metzler-Nolte, N. (2009) ″Four-potential″ ferrocene labeling of PNA oligomers via click chemistry. Bioconjugate Chem. 20, 1578−1586. (46) Köster, S. D., Dittrich, J., Gasser, G., Hüsken, N., Henao Castañeda, I. C., Jios, J. L., Della Védova, C. O., and Metzler-Nolte, N. (2008) Spectroscopic and electrochemical studies of ferrocenyl triazole amino acid and peptide bioconjugates synthesized by click chemistry. Organometallics 27, 6326−6332. (47) Gasser, G., Hüsken, N., Köster, S. D., and Metzler-Nolte, N. (2008) Synthesis of organometallic PNA oligomers by click chemistry. Chem. Commun., 3675−3677. (48) Baldoli, C., Falciola, L., Licandro, E., Maiorana, S., Mussini, P., Ramani, P., Rigamonti, C., and Zinzalla, G. (2004) A new ferrocene conjugate of a tyrosine PNA monomer: synthesis and electrochemical properties. J. Organomet. Chem. 689, 4791−4802. (49) Baldoli, C., Licandro, E., Maiorana, S., Resemini, D., Rigamonti, C., Falciola, L., Longhi, M., and Mussini, P. R. (2005) Electrochemical activity of new ferrocene-labelled PNA monomers to be applied for DNA detection: Effects of the molecular structure and of the solvent. J. Electroanal. Chem. 585, 197−205. (50) Baldoli, C., Rigamonti, C., Maiorana, S., Licandro, E., Falciola, L., and Mussini, P. R. (2006) A new triferrocenyl-tris(hydroxymethyl)aminomethane derivative as a highly sensitive electrochemical marker of biomolecules: application to the labelling of PNA monomers and their electrochemical characterization. Chem. Eur. J. 12, 4091−4100. (51) Gasser, G., and Spiccia, L. (2008) Synthesis of a ferrocenyl uracil PNA monomer for insertion into PNA sequences. J. Org. Chem. 693, 2478−2482. (52) Gasser, G., Belousoff, M. J., Bond, A. M., and Spiccia, L. (2006) Facile synthesis and detailed characterization of a new ferrocenyl uracil peptide nucleic acid monomer. J. Org. Chem. 71, 7565−7573. (53) Baldoli, C., Maiorana, S., Licandro, E., Zinzalla, G., and Perdicchia, D. (2002) Synthesis of chiral chromium tricarbonyl labeled thymine PNA monomers via the Ugi reaction. Org. Lett. 4, 4341− 4344. (54) Maiorana, S., Licandro, E., Perdicchia, D., Baldoli, C., Vandoni, B., Giannini, C., and Salmain, M. (2003) Synthesis of the first chiral PNA monomer labeled with a Fischer-type carbene complex. J. Mol. Catal. A: Chem. 204−205, 165−175. (55) Salmain, M., Licandro, E., Baldoli, C., Maiorana, S., Tran-Huy, H., and Jaouen, G. (2001) Transition metal (’Fischer-type’) carbene complexes as protein labelling reagents. J. Organomet. Chem. 617−618, 376−382. (56) Xavier, C., Giannini, C., Gano, L., Maiorana, S., Alberto, R., and Santos, I. (2008) Synthesis, characterization, and evaluation of a novel 99mTc(CO)3 pyrazolyl conjugate of a peptide nucleic acid sequence. J. Biol. Inorg. Chem. 13, 1335−1344. (57) Baldoli, C., Cerea, P., Giannini, C., Licandro, E., Rigamonti, C., and Maiorana, S. (2005) The metal-conjugated peptide nucleic acid challenge. Synlett, 1984−1994.

(58) Zava, O., Zakeeruddin, S. M., Danelon, C., Vogel, H., Grätzel, M., and Dyson, P. J. (2009) A cytotoxic ruthenium tris(bipyridyl) complex that accumulates at plasma membranes. ChemBioChem 10, 1796−1800. (59) Nielsen, P. E., and Egholm, M. (1999) Peptide Nucleic Acids Protocols and Applications, Horizon Scientific Press, Wymondham, UK. (60) Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M. (1989) Data for Biochemical Research, 3rd ed., Oxford University Press, Oxford, UK. (61) Anne, A., Bouchardon, A., and Moiroux, J. (2003) 3′-Ferrocenelabeled oligonucleotide chains end-tethered to gold electrode surfaces: Novel model systems for exploring flexibility of short DNA using cyclic voltammetry. J. Am. Chem. Soc. 125, 1112−1113. (62) Puglisi, J. D., and Tinoco, J. I. (1989) Absorbance melting curves of RNA. Methods Enzymol. 180, 304−325. (63) Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248−254. (64) Ott, I., Scheffler, H., and Gust, R. (2007) Development of a method for the quantification of the molar gold concentration in tumour cells exposed to gold-containing drugs. ChemMedChem 2, 702−707. (65) Schäfer, S., Ott, I., Gust, R., and Sheldrick, W. S. (2007) Influence of the polypyridyl (pp) ligand size on the DNA binding properties, cytotoxicity and cellular uptake of organoruthenium(II) complexes of the type [(η6-C6Me6)Ru(L)(pp)]n+ [L = Cl, n = 1; L = (NH2)2CS, n = 2]. Eur. J. Inorg. Chem., 3034−3046. (66) Kersebohm, T., Kirin, S. I., and Metzler-Nolte, N. (2006) Insertion of an internal dipeptide into PNA oligomers: Thermal melting studies and further functionalization. Bioorg. Med. Chem. Lett. 16, 2964−2968. (67) Sosniak, A. M., Gasser, G., and Metzler-Nolte, N. (2009) Thermal melting studies of alkyne- and ferrocene-containing PNA bioconjugates. Org. Biomol. Chem. 7, 4992−5000. (68) Gross, A., and Metzler-Nolte, N. (2009) Synthesis and characterisation of a ruthenocenoyl bioconjugate with the cyclic octapeptide octreotate. J. Organomet. Chem. 694, 1185−1188. (69) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595−598. (70) Prins, R., Korswagen, A. R., and Kortbeek, A. G. T. G. (1972) Decomposition of the ferricenium cation by nucleophilic reagents. J. Organomet. Chem. 39, 335−344. (71) Peyret, N., Seneviratne, P. A., Allawi, H. T., and SantaLucia, J., Jr. (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38, 3468−3477. (72) Vessières, A., Top, S., Pigeon, P., Hillard, E. A., Boubeker, L., Spera, D., and Jaouen, G. (2005) Modification of the estrogenic properties of diphenols by the incorporation of ferrocene. Generation of antiproliferative effects in vitro. J. Med. Chem. 48, 3937−3940. (73) Plazuk, D., Vessières, A., Hillard, E. A., Buriez, O., Labbé, E., Pigeon, P., Plamont, M.-A., Amatore, C., Zakrzewski, J., and Jaouen, G. (2009) A [3]ferrocenophane polyphenol showing a remarkable antiproliferative activity on breast and prostate cancer cell lines. J. Med. Chem. 52, 4964−4967. (74) Swarts, J. C., Nafady, A., Roudebush, J. H., Trupia, S., and Geiger, W. E. (2009) One-electron oxidation of ruthenocene: Reactions of the ruthenocenium ion in gentle electrolyte media. Inorg. Chem. 48, 2156−2165. (75) Gross, A., Neukamm, M., and Metzler-Nolte, N. (2011) Synthesis and cytotoxicity of a bimetallic ruthenocene dicobalthexacarbonyl alkyne peptide bioconjugate. Dalton Trans. 40, 1382− 1386. (76) Micallef, L. S., Loughrey, B. T., Healy, P. C., Parsons, P. G., and Williams, M. L. (2011) Mono- and 1,1′-disubstituted organoruthenium cyclopentadiene complexes: synthesis, structural characterization, and antitumoral evaluation. Organometallics 30, 1395−1403. 1773



Article

(77) Ott, I., Schmidt, K., Kircher, B., Schumacher, P., Wiglenda, T., and Gust, R. (2005) Antitumor-active cobalt-alkyne complexes derived from acetylsalicylic acid: Studies on the mode of drug action. J. Med. Chem. 48, 622−629. (78) Dunitz, J. D., Orgel, L. E., and Rich, A. (1956) The crystal structure of ferrocene. Acta Crystallogr. 9, 373−375. (79) Hardgrove, G. L., and Templeton, D. H. (1959) The crystal structure of ruthenocene. Acta Crystallogr. 12, 28−32. (80) Long, N. J. (1998) Metallocenes: an introduction to sandwich complexes, Blackwell Science, Oxford, UK.

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A RuthenoceneâPNA Bioconjugate â Synthesis, Characterization

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