γ-Ferrocenyl Adenosine Triphosphate - American Chemical Society

Jun 22, 2011 - Sanela Martic, Meghan K. Rains, Daniel Freeman, and Heinz-Bernhard Kraatz*. Chemistry Department, The University of Western Ontario, ...
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Use of 50-γ-Ferrocenyl Adenosine Triphosphate (Fc-ATP) Bioconjugates Having Poly(ethylene glycol) Spacers in Kinase-Catalyzed Phosphorylations Sanela Martic, Meghan K. Rains, Daniel Freeman, and Heinz-Bernhard Kraatz* Chemistry Department, The University of Western Ontario, London, Ontario, Canada

bS Supporting Information ABSTRACT:

The 50 -γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2R), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which KM values were determined to be in the 30200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing proteinprotein interactions.

’ INTRODUCTION Protein phosphorylation, catalyzed by protein kinase, is a critical biological event that regulates cellular life and death and has been linked to diseases such as cancer.1,2 A number of analytical methods have been developed to detect and monitor protein kinase activity and to screen for inhibitors of kinases as drug targets in cancer research.313 Monitoring phosphorylation reactions of peptides and proteins is generally achieved by two approaches based on the modified peptide substrates or modified adenosine triphosphate (ATP) analogues. A modification of ATP at the γ-phosphate position by a reporter group, such as a redox or fluorescence label, positions the probe in close proximity to the kinase active site, catalytic pocket, and peptide (protein) substrate to be phosphorylated.1417 By using γ-modified ATP, the phosphorylation-dependent kinasesubstrate cross-linking was recently used to identify phosphorylation sites and active kinases.18 Other γ-substitutions of ATP include biotinylation,19,20 dansylation,21 and thiophosphate22,23 labeling and were used for quantitative determination of kinase activity, identification of peptide (protein) substrates, and screening of kinase inhibitors. An electrochemical strategy was recently developed based on 50 -γferrocenyl phosphate ATP co-substrate (Fc-ATP) to monitor r 2011 American Chemical Society

phosphorylation of surface-bound peptides.13 As depicted in Scheme 1A, the protein kinase-driven Fc-phosphoamide transfer to the Ser, Thr, or Tyr residues of the immobilized peptides resulted in the current response that was subsequently measured. Herein, we explore the utility of new Fc-ATP analogues containing poly(ethylene glycol) linkers between the ferrocene group and the ATP fragment in order to compare the protein kinase promiscuity and co-substrate variability with highly polar co-substrates. By comparing the alkyl and poly(ethylene glycol) spacers of comparable length in Fc-ATP bioconjugates, we electrochemically explored the structural and electronic factors surrounding the kinase-catalyzed reactions, which are less accessible by using unmodified ATP. We decided to synthesize two new polar Fc-poly(ethylene glycol)-ATP conjugates with poly(ethylene glycol) (PEG) spacer -(CH2)2O(CH2)2O(CH2)2 (3) and -(CH2)3O (CH2)2O(CH2)2O(CH2)3 (4) and compare their efficiency as kinase cosubstrates with Fc-alkyl-ATP analogue (5) of Received: May 2, 2011 Revised: June 21, 2011 Published: June 22, 2011 1663

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Scheme 1. Schematic Representation of the Electrochemical Kinase Assays Based on the Fc-ATP Co-Substrate 5 for (A) Phosphorylation of the Surface-Bound Peptidea and (B) Phosphorylation of the Surface-Bound Protein in the Presence of Kinasea; (C) Competitive Solution Fluorescence Assay for Peptide Phosphorylation in the Presence of ATP, Fc-ATP Co-Substrate 5, and Protein Kinaseb

a

Fc-phosphoamide transfer to the surface results in the redox response. b Following the kinase reaction, the subsequent addition of kinase-glo reagent produces light proportional to the leftover ATP amount.

similar spacer length.14 Here, we explore the kinase promiscuity, only with respect to Fc-ATP, and investigate the usefulness of hydrophobic and hydrophilic Fc-ATP co-substrates for peptide phosphorylations by multiple protein kinases. We probe the effects of the polar versus nonpolar Fc-ATP conjugates on four protein kinases and their ability to drive peptide phosphorylation. By electrochemical means, we demonstrate the usefulness of the analytical method for detection of protein phosphorylation as well, as presented in Scheme 1B. The kinase reaction parameters were estimated electrochemically, and the electrochemical trends were compared to the fluorescence kinase assay for the first time (Scheme 1C).

’ EXPERIMETNAL PROCEDURES General Methods. All organic solvents were freshly distilled unless otherwise specified. All reagents were used without further purification. Ferrocene, triethylamine, O-benzotriazole-N,N,N0 , N0 -tetramethyl-uronium-hexafluoro-phosphate (HBTU), hydroxybenzotriazole (HOBt), N,N0 -dicyclohexylcarbodiimide (DCC), and adenosine triphosphate (ATP) were purchased from Sigma Aldrich, while diaminopoly(ethylene glycol) starting material was obtained from TCI America. The peptide substrates ArgArg-Arg-Asp-Asp-Asp-Ser-Asp-Asp-Asp and Arg-Arg-Leu-SerSer-Leu-Arg-Ala, and proteins CK2R, and PKA protein kinases were donated by D. W. Litchfield’s laboratories (The University of Western Ontario, Canada). Src protein kinase and Glu-GlyIle-Tyr-Asp-Val-Pro peptide substrate were obtained from Cell Signaling (New England Biolabs Ltd., Pickering, Ontario). CDK2/Cyclin A kinase and peptide substrate His-His-Ala-SerPro-Arg-Lys were purchased from Enzo Life Sciences. All NMR experiments (1H, 13C, and 31P) were performed on INOVA 400 spectrometer at room temperature. An external reference 85%

H3PO4 was used for all 31P NMR experiments. MALDI-TOF MS spectra were collected on Applied Biosystems 4700 Proteomics Analyzer (Applied Biosystems, USA). All organic solvents were freshly distilled, and the experiments in aqueous conditions were prepared using ultrapure water (18.3 MΩ cm) from Millipore Milli Q system. General Procedure for the Synthesis of the Fc-Poly(ethylene glycol)-Boc 12. To a solution of HBTU (1.36 g, 3.59 mmol), HOBt (0.55 g 3.59 mmol), and triethylamine (0.50 mL, 3.57 mmol) in CH2Cl2 (50 mL) was added a solution of ferrocene monocarboxylic acid (0.69 g 3.01 mmol) in CH2Cl2 (10 mL). The resulting mixture was stirred for 2 h at room temperature and washed with saturated aqueous NaHCO3 (3  50 mL), then 10% citric acid (3  50 mL) and water (3  50 mL). The organic layer was dried over Na2SO4 and evaporated prior to purification. Fc-active ester was purified using flash silica chromatography CH2Cl2/ethyl acetate (90:10% v/v) and isolated in 75% yield. To a solution of Fc-active ester (0.30 g, 0.83 mmol) in CH2Cl2 (30 mL) was added NH2-poly(ethylene glycol)-Boc ligand (0.19 g, 0.79 mmol) in CH2Cl2 (10 mL). The solution was stirred at room temperature for 2 h and then washed with saturated aqueous NaHCO3 anhydrous (3  50 mL), then 10% citric acid (3  50 mL) followed by water (3  50 mL). The organic layer was dried over Na2SO4 and evaporated. The organics were purified using silica chromatography and a CHCl3/MeOH (95:5% v/v) solvent system to afford compounds 1 and 2 as viscous liquids in 86% (0.32 g) and 85% (0.31 g) yield, respectively. Compound 1. 1H NMR (CDCl3, 400 MHz): δ 6.26 (s, 1H, Fc-CO-NH), 5.04 (s, 1H, NH-Boc), 4.69 (s, 2H, Fc), 4.34 (s, 2H, Fc), 4.21 (s, 5H, Fc), 3.66 (s, 4H, OCH2CH2O), 3.62 (m, 2H, CH2), 3.59 (m, 4H, NHCH2, CH2), 3.36 (q, 2H, NHCH2, J = 5.1, 9.9 Hz), 1.46 (s, 9H, Boc). 13C NMR (CDCl3, 400 MHz): δ 156.0 (2C), 79.1, 72.9 (2C), 70.2 (4C), 70.1 (5C), 52.0, 45.9, 1664

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Bioconjugate Chemistry 41.4, 40.3, 31.2, 28.4 (3C). IR (KBr pellet): v (cm1) 3400, 2942, 2924, 1653, 1541, 1457. MS-EI+: m/z (%) 460.1 (100) [M+]. HRMS EI+ m/z (%) calcd 460.1661 for C22H32FeN2O5+, found 460.1666. Compound 2. 1H NMR (CDCl3, 400 MHz): δ 6.55 (s, 1H, Fc-CO-NH), 4.95 (s, 1H, NH-Boc), 4.71 (t, 2H, Fc, J = 1.7 Hz), 4.31 (t, 2H, Fc, J = 1.8 Hz), 4.20 (s, 5H, Fc), 3.753.45 (m, 14H, CH2), 3.23 (q, 2H, NHCH2, J = 5.5, 11.5 Hz), 1.88 (m, 2H), 1.75 (m, 2H), 1.44 (s, 9H, Boc). 13C NMR (CDCl3, 400 MHz): δ 169.9, 156.0, 70.7, 70.5, 70.4, 70.3 (2C), 70.2 (2C), 70.1 (4C), 69.7 (5C), 68.1 (2C), 38.5, 38.3, 29.3, 28.5 (3C). IR (KBr pellet): v (cm1) 3443, 2954, 2924, 2362, 2079, 1636, 1539, 1173. MSEI+: m/z (%) 532.2 (100) [M+]; HRMS EI+: m/z (%) calcd 532.2236 for C26H40FeN2O6+, found 532.2252. General Procedure for Deprotection of Fc-Poly(ethylene glycol)-Boc 12. To a solution of compound 12 (1.21 mmol) in CH2Cl2 (20 mL) was added a solution of trifluoroacetic acid (TFA) (50.1 mmol) in CH2Cl2 (20 mL). The reaction mixture was stirred for 1 h at room temperature. The progress of the reaction was monitored with TLC analysis, and additional TFA was added as a 50% v/v solution in CH2Cl2 as necessary to complete deprotection. The resulting solution was slowly treated with triethylamine (4.21 mL) until a pH of 9 was reached. This solution was washed with water (3  10 mL), and organic layers were dried over Na2SO4, and evaporated to dryness prior to their use. General Procedure for the Synthesis of the Fc-ATP Conjugates 34. The disodium salt of ATP (0.12 g, 0.19 mmol) was equilibrated in 0.1 M triethylammonium bicarbonate (TEAB) buffer (pH 7.4), and passed through a DOWEX ion exchange column. After ion exchange, the volume was reduced with rotary evaporation, and then coevaporated with dry methanol (3  5 mL). The triethylammonium ATP salt was dissolved in dry DMF (5 mL) and stirred at room temperature under an Ar atmosphere. DCC (0.15 g, 0.72 mmol) was added and the reaction mixture was allowed to stir under Ar for 3 h at room temperature to form adenosine metaphosphate (AMP). In the final coupling step, this solution was added to a solution of Fc-poly(ethylene glycol)NH2 in dry methanol (10 mL) and stirred under Ar for 30 min at room temperature. The resulting mixture was then poured into water (20 mL) and a fine precipitate was filtered. The mother liquor was loaded onto a diethylaminoethyl (DEAE)-cellulose column that was pre-equilibrated with 0.1 M TEAB buffer, and flushed with water to remove excess starting ferrocene material. DEAE column was further treated with a gradient 0.10.5 M TEAB buffer to isolate ATP-containing fractions. These fractions were further purified by HPLC as described below. The overall yields after HPLC separation were 8% and 14% for compounds 3 and 4, respectively. Compound 3. 1H NMR (D2O, 400 MHz): δ 8.50 (s, 1H, H8), 8.19 (s, 1H, H2), 6.07 (d, 1H, H1’, J = 6.0 Hz), 4.78 (m, 3H, H2’, Fc), 4.57 (dd, 1H, H30 , J = 3.3, 3.5 Hz), 4.48 (t, 2H, Fc, J = 1.8 Hz), 4.39 (q, 1H, H4’, J = 3.0, 5.7 Hz), 4.254.24 (m, 2H, H50 , H50 ’), 4.24 (s, 5H, Fc), 3.66 (t, 4H, J = 5.1 Hz), 3.56 (t, 2H, NHCH2, J = 5.8 Hz), 3.49 (t, 2H, NHCH2, J = 5.3 Hz), 3.09 (m, 4H). 31P NMR (D2O, 400 MHz): δ 0.45 (d, J = 20.4 Hz), 10.56 (d J = 19.3 Hz), 21.88 (t, J = 19.4 Hz). IR (KBr): ν (cm1) 3300 ̅ 3600 (b, OH), 3444 (b, NH free), 2913 (CH aliphatic), 1 2740, 2678, 1653 cm (s, CdO, amide); MALDI-TOF MS m/z (%): 904.0463 (44) [M+K]+ (exp), 904.0933 (100) [M+K]+ (calcd) for C28H42N7O15P3FeK.

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Compound 4. 1H NMR (D2O, 400 MHz): δ 8.51 (s, 1H, H8),

8.21 (s, 1H, H2), 6.08 (d, 1H, H1’, J = 6.0 Hz), 4.73 (m, 3H, H2’, Fc), 4.53 (t, 1H, H30 , J = 3.4 Hz), 4.45 (t, 2H, Fc, J = 1.9 Hz), 4.36 (q, 1H, H4’, J = 2.6 Hz), 4.254.20 (m, 2H, H50 , H50 ’), 4.21 (s, 5H, Fc), 3.603.50 (m, 4H), 3.49 (t, 2H, NHCH2, J = 6.8 Hz), 3.33 (t, 2H, NHCH2, J = 5.3 Hz), 3.27 (m, 4H), 2.86 (q, 2H, CH2, J = 7.3, 16.2 Hz), 1.83 (q, 2H, CH2, J = 6.5, 12.9 Hz), 1.66 (q, 2H, CH2, J = 6.9, 13.3 Hz). 31P NMR (D2O, 400 MHz): δ 0.18 (d, J = 22.7 Hz), 10.51 (d J = 18.2 Hz), 21.85 (t, J = 19.4 Hz). IR (KBr): ν (cm1) 33003600 (b, OH), 3406 (b, ̅ aliphatic), 2739, 2492, 1648 cm1 (s, NH free), 2933 (CH CdO, amide); MALDI-TOF MS m/z (%): 976.1231 (36) [M+K]+ (exp), 976.1508 (100) [M+K]+ (calcd) for C32H50N7O16P3FeK. HPLC Separation. The resulting mixture was evaporated to dryness and purified using HPLC chromatography (Varian Modular reverse-phase Dynamax Macro-HPLC System C18 (21.4 mm  25 cm)). The flow rate was 15 mL/min, UV detector was set at 254 nm, and a 30 min linear gradient composed of A TEAB (pH 7.4) and B CH3CN as solvent system was used. The mobile phase was composed of 100% A to 97% A over 2 min, from 97% A to 92% A over 2 min, from 92% A to 90% A over 2 min, from 90% A to 85% A over 4 min, at 85% A for 2 min, from 85% A to 88% A over 3 min, from 88% A to 90% A over 3 min, from 90% A to 92% A over 2 min, from 92% A to 96% A over 4 min, and from 96% A to 100% A over 4 min. Electrochemical Studies. All electrochemical studies were performed on a CH instrument potentiostat 660B (Austin, TX). For solution experiments, glassy carbon electrode (surface area = 0.02 cm2) was the working electrode, Ag/AgCl in 3 M KCl was a reference electrode, and Pt wire was an auxiliary electrode. Compounds 1 and 2 were tested in dry acetonitrile solutions at 0.1 mM in the presence of 0.1 M tetrabutylammonium perchlorate (TBAP). Compounds 3 and 4 were tested at 0.1 mM in Milli-Q water containing 0.1 M NaClO4. Surface characterization of biosensor interface was performed using a protein-modified gold electrode (0.02 cm2) as a working electrode, Ag/AgCl 3 M KCl as reference electrode, and Pt wire as an auxiliary electrode. CV electrochemical measurements for surface characterization were carried out in the presence of 1 mM solution of K4[Fe(CN)6] 3 3H2O and K3[Fe(CN)6] 3 3H2O in 10 mM sodium phosphate buffer (pH 7.5) in the presence of 50 mM KNO3. All EIS spectra were collected under the same conditions and at the formal potential of Fe4/-3 redox couple (0.25 V vs Ag/AgCl) at 5 mV amplitude and in the 0.1 Hz to 100 kHz range. All impedance spectra are represented as Nyquist plots with real impedance (Zre) vs imaginary impedance (Zim) values. The experimental data were fitted to an appropriate equivalent circuit by using the ZSimWin 2.0 (EChem software). Preparation of Peptide (Protein)-Modified Gold Electrode. Gold electrodes (surface area 0.02 cm2) were cleaned by etching in piranha solution (3:1% v/v H2SO4/30% H2O2) for 5 min, then rinsed with a copious amount of water, and subsequently polished over alumina slurry (0.3 μm then at 0.05 μm). Next, the electrochemical cleaning by cycling was done in H2SO4 (0.5 M) within the potential range 01.5 V until a stable gold oxidation peak was obtained. Finally, gold electrodes were sonicated in freshly distilled ethanol for 10 min and dried under N2. For film formation, gold electrodes were immersed in lipoic acid N-hydrosuccinimide ester (Lip-NHS) solution (2 mM) in ethanol for 3 days at 5 °C. Electrodes were subsequently rinsed with ethanol and incubated in the selected peptide substrate solution 1665

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Bioconjugate Chemistry (100 μM) or protein solution (100 ng/mL) in dH2O for 1 day at 5 °C. The peptide substrates used were Glu-Gly-Ile-Tyr-AspVal-Pro, His-His-Ala-Ser-Pro-Arg-Lys, Arg-Arg-Arg-Asp-Asp-AspSer-Asp-Asp-Asp, and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala and the protein target was His-tagged pro-caspase-3. Electrodes were further rinsed with water and incubated in ethanolamine solution (100 mM) in ethanol for 1 h. Surface blocking of the electrodes was omitted when protein immobilization was performed. After rinsing with freshly distilled ethanol and drying under N2, the peptide-modified gold electrodes or protein-modified gold electrodes were ready for use. Electrochemical Kinase Assay. For kinase reactions, electrodes were immersed in the kinase reaction buffer and kept at 37 °C in a moist incubation chamber for 2 h. The kinase reaction buffer consisted of the kinase assay buffer specific to the kinase, 400 μM Fc-ATP (compound 35), 1 μg/mL kinase protein (Src, CDK2, and PKA), and 10 ng/mL of CK2R due to limited availability of the given kinase. The kinase buffer recipes used were similar to those suggested by the protein providers, except with the omission of dithiothrietol (DTT). Electrodes were rinsed with 10 mM sodium phosphate buffer (pH 7.5) prior to electrochemical measurements. A typical electrochemical experiment was performed in 10 mM sodium phosphate buffer (pH 7.5) in the presence of modified gold electrode as the working electrode, Ag/AgCl 3 M KCl as reference, and Pt wire as auxiliary electrode. Control experiments were performed by incubating gold electrodes, previously modified with Lip-NHS, into the solution of bovine serum albumin (60 mg/mL) in dH2O. The phosphorylation reactions were performed as described above. The second type of control experiment was carried out on the His-tagged pro-caspase-3 protein modified gold electrodes in the presence of CDK2 protein kinase and in the absence of CK2R. CK2R concentration-dependent kinase assays were carried out at 400 μM compound 5 at following kinase concentrations: 0, 0.1, 0.5, 0.6, 2.5, 5, and 10 ng/mL. Concentration-dependent studies with respect to compound 5 were performed for all four kinases and their respective target peptides at following Fc-ATP concentrations: 5, 10, 25, 50, 75, 100, 200, and 400 μM. These studies were used to determine the KM values for compound 5 with respect to all 4 kinases. Luciferase Kinase Assays. Screening protocol was based on the Kinase-Glo Max luminescent kinase assay.25 Kinase-Glo Max reagent was purchased from SignalChem (USA) and the fluorescence assays were performed as suggested by the provider. A typical reaction mixture consisted of kinase assay buffer specific for a given kinase protein under study, 100 ng/mL of specific protein kinase (CK2R was at 0.5 ng/mL), and 100 μM peptide substrate, at variable ATP concentrations (0, 0.5, 1, 2.5, 5, 10, 25, 35, 50 μM) in the presence or absence of compound 35 (10 μM). The kinase reaction was carried out in a 20 μL reaction volume in a 96-half-well white microtiter plate at 37 °C for 4 h. The microtiter plate was cooled down to RT before adding 20 μL of KinaseGlo Max reagent. After 15 min of further reacting with KinaseGlo Max reagent at room temperature, the luminescence signal was recorded with the PerkinElmer Wallac 1420 Multichannel Counter.

’ RESULTS AND DISCUSSION Synthesis of Hydrophilic Ferrocene Conjugates. N-tertButoxycarbonyl (Boc) protected amino poly(ethylene glycol)

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Scheme 2. Synthesis of the Ferrocene Conjugates 14 and the Structure of Fc-Bioconjugate 5

intermediates were synthesized according to the literature procedure.26 The synthesis of the ferrocene-polyethylene glycol(N-tert-butoxycarbonyl) (Fc-CO-NH-(polyethylene glycol)n-NHBoc) (n = 2 (1), 3 (2)) conjugates was performed in two steps as shown in Scheme 2. First, activated ferrocene-benzotriazole ester was prepared in the presence of HBTU, HOBt, and triethylamine and isolated.27 Subsequently, Boc-protected poly(ethylene glycol) amines were coupled to the Fc-active ester to yield target compounds 1 and 2 in 8090% yield. The compounds were characterized by NMR and mass spectrometry. For the coupling protocol to ATP, the Boc-protected Fc-conjugates were deprotected under acidic conditions, in the presence of an excess trifluoroacetic acid in CH2Cl2. The coupling procedure to ATP was performed as reported in the literature, wherein in situ formed adenosine metaphosphate was reacted with the Fc-poly(ethylene glycol)-NH2 under basic conditions.28,29 The crude reaction mixture was first purified on diethylaminoethyl (DEAE) cellulose column equilibrated with 0.1 M triethylammonium bicarbonate (TEAB) (pH 7.5), in order to remove excess Fc-linker starting material. Additional treatment with 0.10.5 M TEAB afforded the Fc-ATP-containing fractions which were subjected to the reverse-phase HPLC purification. HPLC conditions employed were based on the TEAB/CH3CN solvent system with gradient 100%/0% to 85%/15% to isolate Fc-ATP conjugates 3 and 4 in 8% and 14% yield, respectively. HPLC traces of FcATP bioconjugates 3 and 4 are shown in Figure 1A. Fc-ATP 1666

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Figure 1. (A) HPLC traces of Fc-ATP conjugates 3 and 4. The purification was performed on C18 reverse-phase column, at 10 mL/min flow rate and in TEAB/CH3CN gradient (100% to 85% TEAB). Fc-ATP conjugates are characterized with the retention times of 13.2 (3) and 14.6 min (4) which are higher than that of unmodified ATP (∼6 min). Partial mass spectra of purified compound 3 (B) and 4 (C) showing [M + K]+ molecular ion splitting pattern for experimental (bottom) and theoretical isotope peaks (top).

derivatives were characterized by 1D and 2D NMR and MALDITOF mass spectrometry. Solution Characterization of Ferrocene Conjugates. A distinguishing feature of the 31P{1H}-NMR spectra of the FcATP conjugates 3 and 4 is the characteristic doublet for the γ-P, observed at 0.45 ppm for compound 3 and 0.15 ppm for compound 4 (vs H3PO4 85%), indicative of the resulting Fc-γphosphoamide formation. By contrast, an unmodified ATP exhibits a 31P NMR γ-doublet at approximately 8 ppm. Further characterization of Fc-ATP bioconjugates was performed by MALDI-TOF MS in Figure 1B,C, which shows the molecular ion peak [M+K]+ and isotope pattern for 3 and 4 with the matching theoretical predictions. Solution electrochemical behavior of Fc-linker compounds 1 and 2, presented in Figure 2A, was investigated by means of cyclic voltammetry in CH3CN solution containing TBAP electrolyte. Notably, both compounds exhibit the potential difference, ΔE, in the 5762 mV range, half-wave potential, E1/2, at ∼600 mV and

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peak current ratios, ipa/ipc, at ∼1.1, characteristic of ferrocene compounds. Unlike ATP-free compounds, Fc-bioconjugates 3 and 4 are characterized by a cathodic shift in redox potential to lower values that fall in the 410425 mV range; however, they exhibit ipa/ipc values close to unity and ΔE values in the 4065 mV range (Figure 2B). Comparison of the diffusion coefficients for all four compounds clearly demonstrates the effect of molecule size on its mobility and redox activity in solution, and as expected, Fc-ATP conjugates have smaller D values (∼5  105 cm2/s) than their parent compounds 1 and 2 (∼11  105 cm2/s). Diffusion coefficient values were calculated using the Randles-Sevcic equation.30 Electrochemical and solution data for Fc-conjugates 14 are presented in Table 1. Utility of Fc-ATP Co-Substrates in Peptide Phosphorylation Reactions. This study is aimed at addressing the generality of the Fc-ATP co-substrates and elucidates the effects of the linker polarity in Fc-ATP on four protein kinases under study. Previous studies showed that Fc-ATP conjugates of optimal alkyl linker length are readily consumed in kinase-catalyzed phosphorylation of surface-bound peptide.13,24 However, the effects of the linker variation in Fc-ATP, which includes a more polar poly(ethylene glycol) spacer, on the kinase phosphorylation reaction were unknown. With this goal in mind, we developed and initially investigated compounds 3 and 4 as phosphorylation co-substrates using the general electrochemical kinase assay shown in Scheme 1A. To compare the abilities of polar Fc-ATP compounds 3 and 4 to serve as co-substrates, the aliphatic Fc-ATP analogue 5 was investigated as well, under identical experimental conditions. This allowed for a direct comparison of hydrophobic versus hydrophilic Fc-ATP conjugates while keeping the spacer length relatively constant. The comparative study may reveal more information about the kinase active site and kinase interactions with the peptide substrates. It is also expected that comparative Fc-ATP study will hint toward tolerance of kinases in terms of the structural and electronic effects as they pertain to the co-substrate. Four different kinases were chosen as representative protein kinases that act on Tyr, Ser, or Thr residues. Sarcoma-related (Src) kinase is a protein Tyr kinase that is overexpressed in certain solid cancer tumors.31 Cyclin-dependent kinase (CDK2), in combination with cyclin A, is primarily involved in the cell cycle and division and it phosphorylates Ser residues on the target peptides or proteins.32 While CDK2 is a Pro-directing protein kinase, casein kinase II (CK2R) is an acidophilic kinase and relies on acidic residues as the dominant specificity determinants of peptide and protein substrates.33 CK2R is directly involved in the cellular metabolism and differentiation and phosphorylates Ser/Thr residues in key regulatory proteins.34 Last, protein kinase A (PKA) was chosen as another Ser/Thr kinase that is largely dependent on the presence of cyclic adenosine monophosphate whose binding activates kinase and allows for binding of ATP and subsequent phosphorylation reaction.35 Cyclic voltammetry (CV) and square-wave voltammetry (SWV) were used for electrochemical studies on peptides bound to gold surfaces. These films were prepared by immobilization of the LipNHS on gold, followed by conjugation with the target peptide sequences: Glu-Gly-Ile-Tyr-Asp-Val-Pro (Src), His-His-Ala-SerPro-Arg-Lys (CDK2), Arg-Arg-Arg-Asp-Asp-Asp-Ser-Asp-AspAsp (CK2R), and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (PKA) and finally blocking with ethanolamine, which reacted with any remaining NHS groups. Next, phosphorylation reactions were performed by incubating peptide-modified gold electrodes in the kinase assay buffer 1667

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Figure 2. Cyclic voltammograms of (A) compounds 1 and 2 (0.1 mM in 0.1 M TBAP in CH3CN) and (B) compounds 3 and 4 (0.1 mM in 0.1 M NaClO4 in water).

Table 1. Solution Characterization Data of Fc-conjugates 15a Fc-compound

Rt (min)b

1 2

— —

3

13.2

E1/2 (mV)

ΔE (mV)

ipa/ipc

Dd

— —

600 ( 5 601 ( 5

61 ( 5 57 ( 5

1.1 1.1

12.1 ( 0.13 11.4 ( 0.18

0.45

411 ( 5

61 ( 4

0.9

5.25 ( 0.09

422 ( 3

55 ( 10

1.1

5.77 ( 0.19

424 ( 3

55 ( 10

0.9

4.82 ( 0.32

P NMR (ppm)c

31

10.54 21.88 4

14.6

0.15 10.51 21.82

5e

10.1

0.71 11.40 22.80

a

Electrochemical data for compounds 1 and 2 were obtained at 0.1 mM ligand in CH3CN in the presence of 0.1 M TBAP and the compounds 3 and 4 were measured at 0.1 mM in the presence of 0.1 M NaClO4 as supporting electrolyte. Ag/AgCl as reference electrode, Pt as auxiliary electrode, glassy carbon as working electrode, scan rate = 100 mV/s. Electrochemical data measured by cyclic voltammetry. The reported error was obtained from the triplicate measurements. E1/2 = half-wave potential; ΔE = peak separation; ipa/ipc = peak current ratio. b HPLC conditions: reverse-phase C18 column, solvent gradient: 100% triethylammonium bicarbonate (TEAB), 85% TEAB and 15% CH3CN, 100% TEAB over 30 min. c D2O, 298 K, H3PO4 85% as an external NMR reference. d Diffusion coefficient is given in 105 cm2/s and was determined electrochemically. e Solution data for Fc-conjugate 5 was added as reference.24

containing the appropriate protein kinase (1 μg/mL for all kinases, except for CK2R which was at 10 ng/mL for all peptide assays) and Fc-ATP conjugates 35 (400 μM). Following the kinase-catalyzed phosphorylation reactions and extensive washing with buffer, CVs and SWVs were collected with all the compounds studied (see Supporting Information, Figure SI-1921). Figure 3A,B shows representative CVs and SWVs for Src-catalyzed reactions in the presence of all Fc-ATP conjugates. The largest current densities were observed for the phosphorylation reactions performed with compound 5. The formal redox potential, E°, located at ∼420 V in the case of compound 5 and ∼505 mV in the case of compound 3 suggested the presence of redox active groups on the surface. The current response was directly related to the transfer of ferrocenyl-phosphate group from Fc-ATP cosubstrate to the hydroxyl residues of the surface-bound peptide. Similarly, the electrochemical assays were performed in the presence of other kinases and the compilation of all the electrochemical data is shown in Figure 3C. From Figure 3C, it is evident that the greatest electrochemical signal was observed for CDK2 kinase reactions performed in the presence of hydrophobic compound 5, followed by compounds 4 and 3. In all cases, hydrophobic Fc-ATP conjugate 5 outperformed the polar Fc-ATP compounds

and led to the greatest current density. Relative to each other, compounds 3 and 4 exhibited some difference in their performance depending on the type of kinase protein studied. In the case of Src and CK2R, compound 3 was a better co-substrate, while compound 4 gave the greater signal in the case of CDK2. Given that different kinases exhibit slightly different kinase activities with specific peptide substrates, it is not surprising that the electrochemical signal varies from kinase to kinase protein even with the best Fc-ATP co-substrate, such as 5. However, differences observed between the conjugates for a specific protein kinase can be attributed to the abilities of these compounds to function as co-substrates. Because of the low sensitivity toward polar Fc-ATP compounds, we have investigated kinetic parameters for compound 5 with all four kinases. From the plot of the steady-state current density versus compound 5 concentrations, the LineweaverBurk plot was derived and the KM values were estimated, with respect to the co-substrate, and found to be 30, 150, 90, and 180 μM for Src, CDK2, PKA, and CK2R kinases, respectively. The electrochemical data indicated that the bioconjugate 5 displayed KM values similar to those of ATP, which are typically in the 10100 μM range, depending on the kinase type and the target peptide. 1668

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Figure 3. (A) Cyclic voltammograms and (B) square-wave voltammograms showing current densities of surface-bound peptides following the Src kinase-catalyzed phosphorylation reaction in the presence of compounds 3 (a), 4 (b), and 5 (c). (C) Plot of current densities of surfacebound peptides following the phosphorylation reactions with different kinases (Src, CDK2, CK2R, and PKA) in the presence of compounds 35. Data points are the average of triplicate measurements taken from normalized SWVs.

Finally, we used the solution biochemical luminescent kinaseglo assay, an ATP depletion assay, to investigate the ability of FcATP to act as co-substrates in the presence of unmodified ATP. The fluorescence intensity is directly proportional to the ATP concentration and inversely proportional to the extent of the phosphorylation reaction.25 As presented in Scheme 1C, following the kinase reaction, the greater amount of leftover ATP produces greater fluorescence signal. Initially, the variable ATP concentration experiments were performed with all four kinase proteins and their respective peptide substrates. Next, in a competitive fluorescence assay, all three Fc-ATP conjugates were added at 10 μM in the presence of protein kinases and at variable ATP concentrations (Scheme 1C). The addition of Fc-ATP to ATP assay resulted in the fluorescence increase, as shown in Figure 4A. This observation was not simply due to the addition of Fc-ATP bioconjugates 35, since no fluorescence increase was observed in the kinase assays in the absence of kinase and substrate under identical experimental conditions. Moreover, the leftover ATP in the kinase reaction was directly related to the overall fluorescence. Luminescence increase due to the addition

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of Fc-conjugates 35 was in the 2660% range, depending on the protein kinase as depicted in Figure 4B. The increase in the fluorescence might be due to the ability of compounds 35 to compete with ATP for kinase and subsequent phosphorylation. Hence, an increase in fluorescence is due to the increased leftover ATP concentration. Overall, Fc-ATP conjugates produced a significant increase in the fluorescence intensities for all four kinases, which was similar to the electrochemical data. Discrepancies between the fluorescence and electrochemical assays were potentially due to the state of the peptide substrate, which is free in the solution studies and surface-bound in the electrochemical analysis. However, the electrochemical and fluorescence data indicate that Fc-ATP conjugates are viable substrates for Tyr and a variety of Ser/Thr protein kinases. Moreover, the fluorescence data show that all three Fc-ATP bioconjugates are competitive with respect to ATP and, hence, support the notion that Fc-ATP co-substrates are being utilized by protein kinases. Polar Fc-ATP Co-Substrates in Protein Phosphorylation Reactions. In order to test the compatibility of Fc-ATP cosubstrates with whole protein targets, a kinase-catalyzed phosphorylation of a full-length protein was investigated. As a representative example, His-tagged pro-caspase-3 (dead) (Caspase-3, for short) protein was chosen as the substrate for CK2R protein. Caspases play a major role in the regulation of cell survival,36 and a number of phosphorylated proteins are less susceptible to caspase cleavage.37 Hyperphosphorylation by protein kinases promotes cancerous cell activity through direct interactions with caspase-mediated signaling pathways.38,39 For example, the phosphorylation of pro-caspase-2 by CK2 prevents dimerization and activation.40 In the electrochemical assay, depicted in Scheme 1B, protein Caspase-3 was immobilized on the gold surface in a similar manner to the peptide immobilization. Surface characterization of the biosensor interface was performed by CV and electrochemical impedance spectroscopy (EIS) and in the presence of Fe4/3 redox couple in order to demonstrate the surface coverage and blocking. CVs revealed a reversible redox behavior characteristic for Fe4/3 on bare gold, but a significant reduction in overall current following the protein immobilization. EIS spectra for bare electrodes are characterized by a small resistance component. By contrast, incubation in Lip-NHS solution produces a film resistance in the shape of a semicircle that is magnified when protein was attached. The diameter of the semicircle corresponds to the interfacial resistance at the electrode surface and is quite small in the case of bare Au electrode. The low-frequency loop observed for bare Au surface is due to the diffusion-limited electrochemical process and is lessened upon formation of selfassembled monolayer by Lip-NHS. The large semicircle is evident following the protein binding and suggests the film’s resistance to the ion diffusion. Electrochemical data were fitted to a suitable equivalent circuit that reflects the real electrochemical process. The chosen circuit includes the ohmic resistance, Rs, of the electrolyte solution, the electronic charge transfer resistance, RCT, in parallel with a constant phase element, CPE, which is associated with the double layer and reflects the interface between the assembled film and the electrolyte solution. In addition, the electronic charge transfer resistance is in series with another capacitive component, CPE, that is in parallel with resistance, RX. The RCT value increased from 36 to 1309 Ω cm2 upon formation of Lip-NHS self-assembled monolayer on the gold surface, followed by immobilization of Caspase-3. The phosphorylation reactions were performed for all three Fc-ATP conjugates in the presence of 0.5 ng/mL CK2R protein 1669

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Figure 4. (A) Luminescence intensity versus ATP concentration in the absence (0) and presence of 10 μM compounds 3 (2), 4 (b), and 5 (9) for Src kinase-catalyzed phosphorylation reaction. (B) Plot of the fluorescence intensities for variety of protein kinase phosphorylation reactions in the presence of 50 μM ATP and following the addition of 10 μM Fc-conjugates 35.

Figure 5. Plot of current densities for CK2R-catalyzed phosphorylation of Caspase-3 protein in the presence of (A) Fc-ATP compounds 35 at 0.5 ng/mL kinase, (B) Fc-conjugate 5 at varying CK2R kinase concentrations, and (C) compound 5 at various concentrations. (D) Current densities for the phosphorylations of Caspase-3 and BSA in the presence of 0.5 ng/mL CK2R and phosphorylation of Caspase-3 in the presence of CDK2 kinase. Error bars obtained from triplicate measurements.

kinase. The highest current density, in Figure 5A, was observed after phosphorylation reaction with aliphatic congener Fc-ATP conjugate 5. CVs and SWVs indicated that the efficiency of the ferrocenyl-phosphate group transfer followed the order: compound 5 > 4 > 3 (Figure 5A). The relative order of the hydrophilic compounds was reversed when compared to the phosphorylation of the surface-bound peptide. These differences could be ascribed to many factors including the nature of the substrate to be phosphorylated which are beyond the scope of

this paper and have not been further investigated. We have, however, focused on the most promising compound 5 and investigated the effect of CK2R protein concentration on the extent of phosphorylation reaction and the electrochemical signal. From the plot of current density versus CK2R concentration in Figure 5B, the saturation point at ∼2.5 ng/mL is evident. Fc-ATP concentration studies shown in Figure 5C clearly indicate that the optimal concentration of Fc-conjugate 5 is ∼100 μM. 1670

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Bioconjugate Chemistry We next investigated the selectivity and specificity of protein phosphorylation by using bovine serum albumin (BSA), which is not a kinase substrate, and CK2R protein kinase. Figure 5D indicates a relatively low current density when BSA was used compared to the phosphorylation of the Caspase-3 result. In order to further probe the phosphorylation of Caspase-3 protein, alternative Ser protein kinase, CDK2 was used. While the extent of protein phosphorylation is quite low, judging from the lower current density, the probability of the CDK2-driven phosphorylation can be explained. Caspase-3 is a poor substrate of CDK2, but two Ser residues can potentially be phosphorylated to a lesser extent. As a Pro-directing protein kinase, CDK2 phosphorylation may also overlap with caspase recognition site, which might explain the positive electrochemical response.41 Electrochemical studies suggested that the dramatic differences between the Fc-ATP conjugates might be due to the steric and electronic factors, which surround the phosphorylation event. We have previously established that the optimal linker length in Fc-ATP was necessary to observe Fc-phosphorylation in the electrochemical assay. Since all Fc-ATP conjugates presented here contain relatively long spacers, the steric considerations in the kinase active site are minimal. Despite the fact that the catalytic site of protein kinase, at the γ-phosphate, is shallow and solventexposed, the inherently polar amino acid residues are critical for catalysis. The residues that line the ATP-binding site are hydrophobic by large, but the catalytic site of protein kinase is typically populated with charged amino acid residues such as Lys33 and Asp145, as in case of CDK2/Cyclin A complex.42 The γ-phosphate group of ATP is held together in position by ionic and H-bonding interactions which are critical. Similarly, the catalytic unit in CK2R is lined with Lys68, Asp175, and Glu81 from C-helix.43 Notably, the side chain of Lys168 in the catalytic loop of PKA kinase, in combination with the backbone Ser53 residue, is involved in H-bonding with γ-phosphate, which orients it for a transfer to the substrate.44 In the case of Src kinase, a conserved Lys295 and Asp404 in the activation loop are required for the catalytic action of the kinase, and any changes in the position of the loop regulates the access of the substrate to the active-site cleft and also modulates the phosphorylation.45 In our system, polar poly(ethylene glycol) linkers in Fc-ATP 34 were not optimal for kinase-catalyzed reactions, as indicated by the relatively low current densities. Presumably, the H-bonding interactions of the poly(ethylene glycol) chains in Fc-ATP with the catalytic pocket of the kinase, and most likely with the incoming peptide (protein) substrate, might negatively affect the outcome of the phosphorylation reaction. Since the aliphatic Fcconjugate 5 is highly nonpolar, we propose that the extent of the electrostatic and H-bonding interactions is lessened and hence does not greatly interfere with the phosphorylation process. To identify the exact nature of the interactions between the Fc-ATP conjugates with the protein kinase, we are currently attempting the co-crystallization of protein kinase with Fc-ATP co-substrate.

’ CONCLUSIONS Comparative electrochemical study between polar and nonpolar Fc-ATP conjugates was utilized in peptide (protein) phosphorylation reactions catalyzed by a variety of protein kinases. Electrochemical data reveal that a long hydrophobic Fc-ATP conjugate outperforms the polar Fc-ATP analogues as an efficient kinase co-substrate. Despite the use of a long spacer, which alleviates any steric constraints during the phosphorylation reaction,

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the additional H-bonding and electrostatic interactions make polar Fc-ATP a poor co-substrate. The electronic factors are the major reasons for large differences observed between the Fc-ATP conjugates. We are currently working on crystallizing out protein kinases with Fc-ATP co-substrates in order to fully understand the exact nature of the interactions.

’ ASSOCIATED CONTENT

bS

Supporting Information. Full characterization of all Fcconjugates, solution CVs and SWVs for compounds 14, CVs and SWVs for kinase reactions with compounds 3 and 4, enzyme kinetic studies and EIS table. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: +1-519-661-3022. Tel: +1-519661-2111 ext. 81561.

’ ACKNOWLEDGMENT S.M. thanks the Ontario Ministry of Research and Innovation for a postdoctoral fellowship. This work was funded by the NSERC strategic grants program and the University of Western Ontario. We are grateful to Prof. D. W. Litchfield for CK2R and PKA protein kinases and RRRDDDSDDD and RRLSSLRA peptides. ’ REFERENCES (1) Maning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934. (2) Blume-Jensen, P., and Hunter, T. (2001) Oncogenin kinase signalling. Nature 411, 355–365. (3) Braunwalder, A. F., Yarvood, D. R., Hall, T., Missbach, M., Lipson, K. E., and Sills, M. A. (1996) A solid-phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates. Anal. Biochem. 234, 23–26. (4) Houseman, B. T., Huh, J. H., Kron, S. J., and Mrkisch, M. (2002) Peptide chips for the quantitative evaluation of protein kinase activity. Nat. Biotechnol. 20, 270–274. (5) Shults, M. D., and Imperiali, B. (2003) Versatile fluorescence probes of protein kinase activity. J. Am. Chem. Soc. 125, 14248–14249. (6) Wang, Z., Lee, J., Cossins, A. R., and Brust, M. (2005) Microarray-based detection of protein binding and functionality by gold nanoparticle probes. Anal. Chem. 77, 5770–5774. (7) Wilner, O. I., Guidotti, C., Wieckowska, A., Gill, R., and Willner, I. (2008) Probing kinase activities by electrochemistry, contact-angle measurements and moelcular-force interactions. Chem.—Eur. J. 14, 7774–7781. (8) Green, K. D., and Pflum, M. K. H. (2007) Kinase-catalyzed biotinylation for phosphoprotein detection. J. Am. Chem. Soc. 129, 10–11. (9) Sato, M., Ozawa, T., Yoshida, T., and Umezawa, Y. (1999) A fluorescent indicator for tyrosine phosphorylation-based insulin signalling pathways. Anal. Chem. 71, 3948–3954. (10) Yoshida, T., Sato, M., Ozawa, T., and Umezawa, Y. (2000) An SPR-based screening method for agonist selectivity for insulin signalling pathways based on the binding of phosphotyrosine to its specific binding protein. Anal. Chem. 72, 6–11. (11) Matsuno, H., Furusawa, H., and Okahata, Y. (2004) Kinetic study of phosphorylation-dependent complex formation between the kinase-inducible domain (KID) of CREB and the KIX domain of CBP on a quartz crystal microbalance. Chem.—Eur. J. 10, 6172–6178. 1671

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Bioconjugate Chemistry (12) Kerman, K., Vestergaard, N., and Tamiya, E. (2007) Label-free electrical sensing of small molecule inhibition on tyrosine phosphorylation. Anal. Chem. 79, 6881–6885. (13) Song, H., Kerman, K., and Kraatz, H. B. (2008) Electrochemical detection of kinase-catalyzed phosphorylation using ferrocene-conjugates ATP. Chem. Commun. 502–504. (14) Parang, K., Kohn, J. A., Saldanha, A., and Cole, P. A. (2002) Development of photo-crosslinking reagents for protein kinase-substrate interactions. FEBS Lett. 520, 156–160. (15) Lee, S. E., Elphick, L. M., Anderson, A., Bonnac, L., Child, E. S., Mann, D. J., and Gouverneur, V. (2009) Synthesis and reactivity of novel γ-phosphate modified ATP analogues. Bioorg. Med. Chem. Lett. 19, 3804–3807. (16) Elphick, L. M., Lee, S. E., Gouverneur, V., and Mann, D. J. (2007) Using chemical genetics and ATP analogues to dissect protein kinase function. Chem. Biol. 2, 299–314. (17) Freeman, R., Finder, T., Gill, R., and Willner, I. (2010) Probing protein kinase (CK2) and alkaline phosphatase with CdSe/ZnS quantum dots. Nano Lett. 10, 2192–2196. (18) Suwal, S., and Pflum, M. K. H. (2010) Phosphorylation-dependent kinase-susbtrate cross-linking. Angew. Che. Int. Ed. 49, 1627–1630. (19) Green, K. D., and Pflum, M. K. (2007) Kinase catalyzed biotinylation for phosphoprotein detection. J. Am. Chem. Soc. 129, 10–11. (20) Wang, Z., Levy, R., Fernig, D. G., and Brust, M. (2006) Kinasecatalyzed modification of gold nanoparticles: a new approach to colorimetric kinase activity screening. J. Am. Chem. Soc. 128, 2214–2215. (21) Green, K. D., and Pflum, K. H. (2009) Exploring kinase cosubstrate promiscuity: monitoring kinase activity through dansylation. ChemBioChem 10, 234–237. (22) Eckstein, F. (1985) Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367–402. (23) Allen, J. J., Lazerwith, S. E., and Shokat, K. M. (2005) Bioorthogonal affinity purification of direct kinase substrates. J. Am. Chem. Soc. 127, 5288–5289. (24) Martic, S., Labib, M., Freeman, D., and Kraatz, H. B. Probing the role of the linker in Fc-ATP conjugates: monitoring protein kinasecatalyzed phosphorylations electrochemically. Chem.—Eur. J. 2011, 17, 67446752. (25) Koresawa, M., and Okabe, T. (2004) High-throughput screening with quantitation of ATP consumption: A universal non-radioisotope, homogenenous assay for protein kinase. Assay Drug Dev. Technol. 2, 153–160. (26) N’Da, D. D, and Neuse, E. W. (2006) Polyamidoamines as drug carriers: synthesis of polymers featuring extrachain-type primary amino groups as drug-anchoring sites. S. Afr. J. Chem. 59, 65–70. (27) Carpino, L. A. (1993) 1-Hydroxy-7azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397–4398. (28) Knorre, D. G., Kurbatov, V. A., and Samukov, V. V. (1976) General method for the synthesis fo ATP gamma-derivatives. FEBS Lett. 70, 105–108. (29) Suto, R. K., Whalen, M. A., Bender, B. R., and Finke, R. G. (1998) Synthesis of γ-phosphate-linked nucleoside affinity chromatography resins for protein purification, including ribonucleoside triphosphate reductase. Nucleosides, Nuleotides Nucleic Acids 17, 1453–1471. (30) Laviron, E. (1979) General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems J. Electroanal. Chem. Interfacial Electrochem. 101, 19–28. (31) Slack-Davis, J., Da Silva, J. O., and Parson, S. J. (2010) LKB1 and Src: antagonistic regulators of tumor growth and metastasis. Cancer Cell 17, 527–529. (32) Malumbres, N., and Barbacid, M. (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. 9, 153–166. (33) Duncan, J. S., Turowec, J. P., Vilk, G., Li, S. S. C., Gloor, G. B., and Litchfield, D. W. (2010) Regulation of cell proliferation and survival: Convergence of protein kinases and caspases. Biochim. Biophys. Acta 1804, 505–510. (34) Litchfield, D. (2003) Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem. J. 369, 1–15.

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(35) Wu, K. J., Mattioli, M., Morse, H. C., and Dalla-Favera, R. (2002) c-MYC activates protein kinase A (PKA) by direct transcriptional activation fo the PKA catalytic subunit beta (PKA-Cβ) gene. Oncogene 21, 7872–7882. (36) McStay, G. P., Salvensen, G. S., and Green, D. R. (2008) Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 15, 322–331. (37) Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L., Antonsson, B., and Martinou, J. C. (2001) Phosphorylation of bid by casein kinase I and II regulates its cleavage by caspase 8. Mol. Cell 8, 601–611. (38) Okun, I., Balakin, K. V., Tkachenko, S. E., and Ivachtchenko, A. V. (2008) Caspase activity modulators as anticancer agenets. Anticancer Agents Med. Chem. 8, 322–341. (39) Torres, J., Rodriguez, M. P., Myers, M., Valiente, M., Graves, J. D., Tonks, N. K., and Pulido, R. (2003) Phosphorylation-regulated cleavage of the tumour suppressor PTEN by caspase-3: implications for the control of protein stability and PETN-protein interactions. J. Biol. Chem. 278, 30652–30660. (40) Li, P. F., Li, J., Muller, E. C., Otto, A., Dietz, R., and Harsdorf, R. (2002) Phosphorylation by protien kinaseCK2: a singling switch for the caspase-inhibiting protien ARC. Mol. Cell 10, 247–258. (41) Schweigreiter, R., Stasyk, T., Contarini, S., Frauscher, S., Oertle, T., Klimaschewski, L. A., Huber, C. E., and Bandtlow, C. E. (2007) Phosphorylation-regulated cleavage of the reticulon protein Nogo-B by caspase-7 at a noncanonical recognition site. Proteomics 7, 4457–5567. (42) De Bondt, H. L., Rosenblatt, J., Jancarik, J., Jones, H. D., Morgan, D. O., and Kim, S. H. (1993) Crystal structure of cyclindependent kinase 2. Nature 363, 595–602. (43) Mazzorana, M., Pinna, L. A., and Battistutta, R. (2008) A structural insight into CK2 inhibition. Mol. Cell. Biochem. 316, 57–62. (44) Taylor, S. S., Yang, J., Wu, J., Haste, N. M., Radzio-Andzelm, E., and Anand, G. (2004) PKA: a portrait of protein kinase dynamics. Biochim. Biophys. Acta 1697, 259–269. (45) Williams, J. C., Weijland, A., Gonfloni, S., Thompson, A., Courtneidge, S. A., Superti-Furga, G., and Wierenga, R. K. (1997) The 2.35 Å crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions. J. Mol. Biol. 274, 757–775.

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