Closer to nature: an ATP-driven bioinspired catalytic oxidation process

Transcription

1 Closer to nature: an ATP-driven bioinspired catalytic oxidation process Loic Stefan, a Damien Duret, b Nicolas Spinelli, b Eric Defrancq b and David Monchaud* a a Institut de Chimie Moléculaire de l Université de Bourgogne (ICMUB), CNRS UMR6302, 9 Avenue Alain Savary, Dijon, France. david.monchaud@u-bourgogne.fr; b Département de Chimie Moléculaire, Université Joseph Fourier, CNRS UMR5250, 570 Rue de la chimie, Grenoble, France. 1 Materials and chemicals.. Page 1 2 DNAzyme and TASQzyme experiments Page 2 3 Control experiments carried out with 22AG/hemin... Page 3 4 UV-Vis titrations. Page 4 5 Investigation of the ph impact Page 6 6 DNAzyme experiments with ATP at ph 4.8 and 7.2. Page Materials and chemicals All DNAzyme UV-Vis experiments were carried out in 1mL-quartz cuvettes (Starna) on a JASCO V630Bio spectrophotometer with a thermally controlled six-cell holded. All chemicals were purchased from Sigma-Aldrich and used without purification (including hemin (iron(iii) protoporphyrin chloride, from bovine), ATP (Adenosine 5 -triphosphate) and ABTS (2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))). DOTASQ, RAFT-G4 and FeTPPS (iron(iii) meso-tetra(4-sulfonatophenyl)porphyrin chloride) were synthesized according to previously reported procedures: for FeTPPS see a) L. Stefan, H.-J. Xu, C. P. Gros, F. Denat and D. Monchaud, Chem. Eur. J., 2011, 17, 10857, b) Z. Dong, P. J. Scammells, J. Org. Chem., 2007, 72, 9881, c) I. Nicolas, P. Le Maux, G. Simonneaux, Tetrahedron Let. 2008, 49, 5793; for DOTASQ see L. Stefan, A. Guédin, S. 1

2 Amrane, N. Smith, F. Denat, J.-L. Mergny and D. Monchaud, Chem. Commun., 2011, 47, 4992; and for RAFT-G4 see P. Murat, B. Gennaro, J. Garcia, N. Spinelli, P. Dumy and E. Defrancq, Chem. Eur. J., 2011, 17, DNAzyme and TASQzyme experiments a with hemin as co-catalyst All the experiments were carried out at 25 C with Caco.KTD buffer, comprised of 10 mm lithium cacodylate buffer (ph 7.2) plus 10mM KCl / 90mM LiCl, 0.05% Triton X-100 and 0.1% DMSO. Stock solutions were ABTS (20mM in water), hemin (100µM in DMSO), H 2 O 2 (60mM in water) and DOTASQ or RAFT-G4 (1mM in DMSO). Experimental conditions were defined as: ABTS (2mM), hemin (1µM), H 2 O 2 (0.6mM) and DOTASQ or RAFT-G4 (from 0 to 100µM). b with FeTPPS as co-catalyst All the experiments were carried out at 25 C with Caco.K buffer, comprised of 10mM lithium cacodylate buffer (ph 7.2) plus 10mM KCl / 90mM LiCl. Stock solutions were ABTS (20mM in water), FeTPPS (100µM in water), H 2 O 2 (60mM in water) and DOTASQ or RAFT-G4 (1mM in DMSO). Experimental conditions were defined as: ABTS (2mM), FeTPPS (1µM), H 2 O 2 (0.6mM) with or without RAFT-G4 (50µM). The RAFT-G4 and FeTPPS mixture in Caco.K buffer were stirred at room temperature for 30min, then ABTS were added and the DNAzyme reaction was thus initialized by the addition of H 2 O 2. 2

3 RAFT-G4/hemin RAFT-G4/FeTPPS Concentration (µm) V 0 (nm.min -1 ) k cat (h -1 ) V 0 (nm.min -1 ) k cat (h -1 ) c with hemin as co-catalyst in presence of ATP All the experiments were carried out at 25 C with Caco.KTD buffer, comprised of 10 mm lithium cacodylate buffer (ph 7.2) plus 10mM KCl / 90mM LiCl, 0.05% Triton X-100 and 0.1% DMSO. Stock solutions were ABTS (50mM in water), hemin (100µM in DMSO), ATP (20mM in Caco.KTD buffer), H 2 O 2 (60mM in water) and DOTASQ or RAFT-G4 (1mM in DMSO). Experimental conditions were defined as: ABTS (5mM), hemin (1µM), ATP (10mM), H 2 O 2 (6mM) and DOTASQ or RAFT-G4 (from 0 to 100µM). The DOTASQ or RAFT-G4, hemin and/or ATP mixtures in Caco.KTD buffer were stirred at room temperature for 30min, then ABTS were added and the DNAzyme reactions were thus initialized by the addition of H 2 O 2. d - Data treatment The characteristic UV-Vis signal of ABTS of each experiment (recorded every 60 seconds at 420nm) was plotted as a function of time with OriginPro 8 software (OriginLab Corp. Northampton, MA, USA). Raw data were firstly subtracted from the control experiment (carried out in the same conditions without pre-catalyst, i.e. DOTASQ or RAFT-G4) and, for sake of comparison, subsequently zeroed at their very first point to obtain the zeroed data. For V 0 determination, the absorbance at the very first four points was plotted as a function of time and linearly fitted to access the slope of this linear function, which corresponds to V 0 (in Abs.min -1 ) subsequently converted in µm.min -1 via the Beer-Lambert law: Abs = ε.l.c, using ε = M -1.cm -1 (for ABTS + ). Furthermore, the catalytic constants k cat (min -1 ) were determined by divided the initial rate V 0 (in µm.min -1 ) by the pre-catalyst concentration (i.e. DOTASQ or RAFT-G4, in µm), finally converted in h -1 for more clarity. 3 Control experiments carried out with 22AG/hemin a without ATP 22AG ( 5 AG 3 (T 2 AG 3 ) 3 3 ) is a quadruplex-forming sequence that has been thoroughly studied in our previous investigations (Chem. Eur. J., 2011, 17, & Nucleic Acids Res., 2012, 40, 8759). The experiments were carried out at 25 C with Caco.K buffer, comprised of 10 3

4 mm lithium cacodylate buffer (ph 7.2) plus 10mM KCl / 90mM LiCl. Stock solutions were: ABTS (20mM in water), hemin (100µM in DMSO), H 2 O 2 (60mM in water), 22AG (269µM in DMSO). Experimental conditions were defined as: ABTS (2mM), hemin (1µM), H 2 O 2 (0.6mM) and 22AG (1µM). b with ATP The experiments were carried out at 25 C with Caco.K buffer, comprised of 10 mm lithium cacodylate buffer (ph 7.2) plus 10mM KCl / 90mM LiCl. Stock solutions were ABTS (50mM in water), hemin (100µM in DMSO), H 2 O 2 (600mM in water), 22AG (269µM in DMSO) and ATP (20mM in Caco.K buffer). Experimental conditions were defined as: ABTS (5mM), hemin (1µM), H 2 O 2 (6mM), 22AG (1µM) and ATP (10mM). c results Using the same data treatment than above (see 2d), following graph and V 0 were obtained: 22AG/hemin 22AG/hemin + ATP (10mM) Concentration (µm) V 0 (nm.min -1 ) k cat (h -1 ) V 0 (nm.min -1 ) k cat (h -1 ) UV-Vis titrations a Studies of the stability of hemin in presence of H 2 O 2 with or without ATP The capability of ATP to protect hemin against H 2 O 2 -degradation was studied via a first series of UV-Vis titrations: the experiments were carried out in 1mL-quartz cuvettes in Caco.KTD 4

5 buffer with 10µM hemin in presence or absence of 6mM H 2 O 2 and/or 10mM ATP, using the following stock solutions: hemin (1mM in DMSO), H 2 O 2 (600mM in water) and ATP (20mM in Caco.K buffer). UV-Vis spectra were recorded in nm range before (t=0min) and after the H 2 O 2 addition (t=2min, 10min, 20min, 30min, 45min and 60min). Quartz cuvettes were stirred at room temperature between each record. Several conclusions can be drawn from that first series of results: a) Firstly, as seen in the upper panels, while hemin is mainly disaggregated in the experimental conditions (λ max = 394nm, left panel), ATP protects it favoring its aggregated state (λ max = 363nm, right panel). b) Secondly, the kinetics of hemin degradation strongly depends on its aggregation state: as seen in the left panels, the disaggregated hemin is rapidly degraded while its aggregated state (right panels) is more robust in the experimental conditions. ATP thus clearly protects hemin against H 2 O 2 -mediated degradation. 5

6 b Effect of TASQ in the hemin aggregation/disaggregation To study the impact of RAFT-G4 and DOTASQ on hemin, UV-Vis experiments were carried out in a 96-well plate in Caco.K buffer with 10µM hemin in presence or absence of 50µM RAFT-G4 and DOTASQ, using the following stock solutions: hemin (1mM in DMSO) and RAFT-G4 and DOTASQ (both 1mM in DMSO). The final volume per well was 200µL. UV-Vis spectra were recorded in the nm range. Both TASQ interact with hemin as judged by the modification of the UV-Vis spectra: hemin alone (black curve) is mainly aggregated in the experimental conditions (λ max = 363nm), and upon interaction with TASQ (either RAFT-G4 (yellow curve) or DOTASQ (red curve)) the disaggregated hemin (λ max = 394nm) appears. These results thus indicate that both TASQ are efficient pre-catalysts since they favor the disaggregated hemin that is known to be the catalytically competent form. 5 Investigation of the ph impact a on the hemin aggregation/disaggregation The predominant form of hemin in solution (i.e. aggregated or disaggregated), a key point of its DNAzyme activity, is modulated by the ph of the solution. The experiments were carried out in 1mL-quartz cuvettes with 2µM hemin in 16 different Caco.K solutions buffered at ph=1.92, 2.95, 3.44, 3.81, 4.27, 4.55, 4.95, 5.51, 6.04, 6.53, 7.24, 7.99, 9.05, 9.95, and 11.92; UV-Vis spectra were recorded in the nm range. Data were zeroed at 800nm (for sake of clarity, only the most significant curves are shown). 6

7 As illustrated by this titration, the influence of the ph is very important: hemin is mainly found under its aggregated form up to ph 7.2 (λ max = 363nm); however, the intensity of the signals tends to demonstrate that hemin is fairly soluble only over ph 5. The disaggregated form of hemin (λ max = 394nm) progressively appears to be the major form beyond ph 9. b Evolution of initial rates V 0 as a function of ph Because of the change in the aggregation states of hemin at different ph, its impact on the initial rate was evaluated via DNAzyme experiments using the previous studied quadruplexforming sequence 22AG. The experiments were carried out in a 96-well plate in 16 different Caco.K buffers (from ph 1.92 to 11.97) in the presence of 1µM 22AG, 1µM hemin, 2mM ABTS and 0.6mM H 2 O 2. Stock solutions were the following: 22AG (269µM in Caco.K), hemin (100µM in DMSO), ABTS (40mM in water) and H 2 O 2 (6mM in water) and the final volume per well was 200µL. According to the previously reported procedure, 22AG was mixed with hemin in buffer and stirred at room temperature for 10min. For sake of comparison, each reaction was simultaneously realized without 22AG (i.e. the catalytic activity of hemin alone, named blank ). Then, ABTS was added and the reaction starts after H 2 O 2 addition. The characteristic absorbance of ABTS + was monitored at 420nm for 160min each 20s. Results are mean of three experiments and zeroed absorbance, defined as the absorbance of 22AG subtracted of the corresponding blank (at the same ph) and zeroed at the very first point, was plotted as a function of time. The initial rates (V 0 in Abs.min -1 ) were determined by fitting as a linear function the 5 very first points (from 0, 0.3, 0.6, 0.9; and 1.2min) and obtained V 0 were subsequently plotted as a function of ph. 7

8 N.B.: due to the non-determined optical length of the solutions in well, the conversion in nm.min -1 using the Beer-Lambert law is not possible herein. ph V 0 (10-3 Abs.min -1 ) ph V 0 (10-3 Abs.min -1 ) These results clearly indicate that, contrarily to what was reported by D. Sen et al. (P. Travascio, Y. Li and D. Sen, Chem. Biol., 1998, 5, 505), ph between 4 and 7 provide the better catalysis, which is optimal at ph DNAzyme experiments with ATP at ph 4.8 and 7.2. The preparation of a 10mM ATP in Caco.K buffer (ph 7.2) surprisingly leads to a strong ph drop (shifting to 4.8), defined as better catalytic conditions (see above). To confirm the actual role of ATP whatever the ph solution, a series of experiments were carried out with four different buffers: classical Caco.K (ph 7.2), acidic Caco.K (ph 4.8), and the equivalent ones comprised of 10mM ATP: ATP.Caco.K (ph 7.2) and ATP.Caco.K (ph 4.8). Thus, in 1mL- 8

9 quartz cuvettes, experiments were carried out with or without 1µM 22AG, 1µM hemin, 2mM ABTS and 0.6mM H 2 O 2, using the four different buffer conditions. Stock solutions were: 22AG (269µM in Caco.K), hemin (100µM in DMSO), ABTS (20mM in water) and H 2 O 2 (60mM in water). Results were treated according to the previous procedure and absorbance values were plotted as a function of time. The initial rates (V 0 in Abs.min -1 ) were similarly determined by fitting as a linear function the 5 very first points and converted as nm.min -1. As seen the figure below, the catalysis is (as expected) more efficient in solution at ph 4.8 (square motifs) than at ph 7.2 (triangle motifs). However, at both ph, ATP (blue lines) still enhances the catalysis as compared to ATP-less conditions (red lines). ph [ATP] (mm) V 0 (nm.min -1 ) These results thus indicate that even if the ATP-mediated acidification of the reaction mixture indeed influences strongly the catalytic efficiency of the DNAzyme system, it does not solely account for the observed positive ATP effect, but rather contributes to that boost alongside the three previously postulated ATP roles (H 2 O 2 -activator, electron-transfer energizer and reaction partners protector, cf. Nucleic Acids Res., 2012, 40, 8759). 9