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EPR Characterization of Cu(II) Complexes of PAMAM-Py Dendrimers for Biocatalysis in the Absence and Presence of Reducing Agents and a Spin Trap Yi-Hsuan Tang, Michela Cangiotti, Chai-Lin Kao, and Maria Francesca Ottaviani J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b09464 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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The Journal of Physical Chemistry

EPR Characterization of Cu(II) Complexes of PAMAM-Py Dendrimers for Biocatalysis in the Absence and Presence of Reducing Agents and a Spin Trap Yi-Hsuan Tang§, Michela Cangiotti‡, Chai-Lin Kao*§,†,#, Maria Francesca Ottaviani*‡

§

Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, 100 Shih-Chuan

1st Road, Kaohsiung 80708, Taiwan ‡

Department of Pure and Applied Sciences, University of Urbino, Via Ca’ Le Suore 2/4, 61029

Urbino, Italy †

Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708,

Taiwan #

Department of Chemistry, National Sun Yat-sen University, 70 Lienhai Rd., Kaohsiung 80424,

Taiwan

*Corresponding Authors: Maria Francesca Ottaviani, Tel. +39-0722304320, [email protected]; Chai-Lin Kao, Tel. +886-7-3121101, [email protected]

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Abstract Polyamidoamine (PAMAM) dendrimers at different generations (from G2 to G6) were functionalized with pyridine (Py) groups at the external surface, and their complexation behaviour with Cu(II) at increasing molar ratios between the ions and the Py groups was analysed in the absence and presence of reducing agents and a spin trap. These Cu(II)-dendrimer complexes may be used as antitumor and antiamyloidogenesis drugs, similarly to other Cu(II)-dendrimer complexes, and as biocatalysts. Indeed, they have revealed to selectively catalyse molecular oxygen reduction to generate reactive oxygen species (ROS). A computer-aided electron paramagnetic resonance (EPR) study of these complexes allowed us to identify different complexes by increasing the Cu(II)/Py molar ratio for the different generations. Binuclear EPR-silent complexes were formed at the highest generations. The differentlycomplexed Cu(II) ions showed a different capability to be reduced, starting from the most exposed at the dendrimer surface bearing a stable Cu(II)-Py2 coordination. Cu(II)-G5 showed peculiar structural properties which probably favoured its activity as biocatalyst. The spin trap was able to capture hydroxyl radicals, which became clearly EPR visible after all Cu(II) ions were reduced to Cu(I). This method may be used as a platform to study interactions of Cu(II) in nano-sized macromolecules for biomedical purposes, mainly in biocatalysis involving redox reactions and formation of ROS.

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1 Introduction

The interaction/complexation between ligands and metallic ions is fascinating and interesting due to the involvement of the complexes in several biological and industrial processes. Such interactions have been intensively studied in case of small molecular ligands. On the contrary, nano-sized macromolecules often provide different interacting sites, which interact with the ions in various ways and may also host a massive amount of metallic ions, showing a different behavior with respect to small molecular ligands. However, the investigation regarding nano-sized macromolecular ligands is still rare until today. One of the reasons is lack of suitable models due to the difficulty of obtaining pure compounds. Among various macromolecules, dendrimers are nanoparticles with unified size and structure [1-8]. Dendrimers possess several equivalent interacting sites available for ion complexation both internally and peripherally. Dendrimers could be produced through iterative incorporation of building blocks. Therefore, their size and structure could be well regulated. Their internal and peripheral groups could be designed and modulated to get a well-defined reactivity that, because of the nano-sized architecture, may be combined in a limited space to display a large affinity for transition metal ions. Dendrimers are, therefore, macromolecular nanometer-scale devices with chemical properties which involve transition metal ions and can be modulated by the macromolecular architecture. Their internal/external chemical structure may be constructed to improve complexes stability in function of different applications. The most interesting applications for biomedical purposes were revealed by recent studies which demonstrated that dendrimer-Cu(II) complexation significantly improved the antitumor activity of selected dendrimers [9-13], and similarly may be used to prevent amyloid fibril formation responsible of Alzheimer diseases [14-15]. Recently, a family of metallodendrimers based on Cu(II) ions and polyamidoamine (PAMAM) dendrimers was found to work as biocatalysts by selectively reducing molecular oxygen to generate 3 ACS Paragon Plus Environment


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superoxide anion radical [16]. In addition to their characteristic catalytic ability, the interaction among coordinated Cu(II) ions are unique. In lower generations of metallodendrimers, each Cu(II) ion individually catalyzed molecular oxygen reduction. In contrast, the active center in 5th and 6th generation dendrimers contained bicopper complexes [17]. These metallodendrimers not only have promising applications for biomedical uses, like anticancer and antiamyloidogenesis drugs and biocatalysts, but also constitute an excellent model and a platform to study interactions of Cu(II) in nano-sized macromolecules. This study analyzes the complexation behavior of Cu(II) with PAMAM dendrimers functionalized with pyridine groups at the external surface (PAMAM-Py) from generation 2 to generation 6 (simply termed G2, G3, G4, G5 and G6), at different molar ratios between the ions and the dendrimer surface sites. Figure 1 shows the structure of generation 2 PAMAM-Py, termed G2.

Figure 1 Structure of the G2-PAMAM-(Py)28 dendrimer 4 ACS Paragon Plus Environment

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The complexation was studied by analyzing the electron paramagnetic resonance (EPR) spectra. The EPR technique already demonstrated to be a powerful tool to obtain information on the complexation behavior of dendrimers in the presence of Cu(II) also working as probe of the interacting ability and structural flexibility of these nanoparticles [18-24]. The Cu(II)-dendrimer complexes were studied in the absence and presence of two reducing agents, dithiothreitol (DTT) and ascorbic acid (AA), to analyze the reduction mechanism of dendrimer-complexed Cu(II) ions to Cu(I). Cu(I) is EPR silent in the experimental conditions of the EPR analysis. A spin trap, N-tert-butyl-α-phenylnitrone (PBN), was added to the solutions to investigate the formation of transient radical species (mainly ROS) derived from the reduction process. PBN covalently binds to the transient radical and becomes a relatively stable nitroxide radical, allowing the identification of the transient radical species.

2 Experimental methods

2.1 Materials

Unless otherwise specified, the chemicals were purchased by Sigma-Aldrich Corporation, St. Louis, MO, USA.

2.2 Synthesis and Characterization of the dendrimers

The preparation of the dendrimers followed the following procedures [16]:

2.2.1 (G:2)-dendri-PAMAM-(pyridine)29

To the solution of (G:2)-dendri-PAMAM-(NH2)16 (103 mg, 0.03 mmol) in DMF (20 mL) was added 2pyridinecarboxaldehyde (680 mg, 6.35 mmol) and NaBH(OAc)3 (1000 mg, 4.72 mmol) at rt. under nitrogen. The resulting solution was added to NaBH(OAc)3 (500 mg, 2.36 mmol) and pyridine-25 ACS Paragon Plus Environment


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carboxaldehyde (499 mg, 4.67 mmol) every day until no positive result from nihydrin test. Excess reagents was removed by dialysis. (3500 MW cutoff). After (152 mg, 86 %).1H-NMR (400 MHz, D2O: 8.64 (d, J = 5.6 Hz, 29 H), 8.44 (t, J = 8 Hz, 29 H), 7.97 (d, J = 8 Hz, 29 H), 7.86 (t, J = 6.4 Hz, 29 H), 4.21 (s, 58 H), 3.52~3.28 (m, 146 H), 2.79~2.69(m, 82 H) Mass (MALDI, m/z) calculated M: 668.3. (M + 3K+ + 6H+); Found: 668.3.

2.2.2 (G:3)-dendri -PAMAM-(pyridine)53 The procedure used to synthesize G2 which was also applied to synthesize G3 (196 mg, 81%). 1H-NMR (400 MHz, D2O) : 8.69 (d, J = 5.6 Hz, 53 H), 8.51 (t, J = 8 Hz, 53 H), 8.03 (d, J = 8.0 Hz, 53 H), 7.92 (t, J = 6.4 Hz, 53 H), 4.27 (s, 104 H), 3.58~3.35 (m, 318 H), 2.85~2.75 (m, 166 H). Mass (MALDI, m/z) calculated M: 592.6 (M + 3Na+ + K+ + 16H+ )/20; 622.9 (M + 4Na+ + 15H+ )/19: 1341.6(M + 9K+ )/9: Found: 592.6; 623.2; 1340.9.

2.2.3 (G:4)-dendri-PAMAM-(pyridine)109 The procedure used to synthesize G2 which was also applied to synthesized G4 (185 mg, 79%). 1HNMR (400 MHz, D2O) : 8.66 (d, J = 5.6 Hz, 109 H), 8.45 (t, J = 8 Hz, 109 H), 7.98 (d, J = 8.0 Hz, 109 H), 7.88 (t, J = 7.2 Hz, 109 H), 4.23 (s, 218 H), 3.55~3.29 (m, 649 H), 2.80~2.71 (m, 354H). Mass (MALDI, m/z) calculated M: 623.6 (M + 4K+ + 35H+)/39, 696.7 (M + 5Na+ + 3K+ + 27H+ )/35, 755.2. (M + 32H+)/32; Found: 623.5; 696.9; 754.3.


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2.2.4 (G:5)-dendri-PAMAM-(pyridine)218 The procedure used to synthesize G2 which was also applied to synthesized G5 (178 mg, 81%). 1HNMR (400 MHz, D2O) : 8.69 (d, J = 5.6 Hz, 218 H), 8.49 (t, J = 7.6 Hz, 234 H), 8.02 (d, J = 8.0 Hz, 218 H), 7.91 (t, J = 7.2 Hz, 218 H), 4.26 (s, 436 H), 3.57~3.34 (m, 1278 H), 2.84~2.75 (m, 728 H). Mass (MALDI, m/z) calculated M: 625.9 (M + 2K+ + 76H+ )/78, 1449.0 (M + 20Na+ + 4K+ + 10H+ )/34, Found: 625.8; 1449.1;

2.2.5 (G:6)-dendri -PAMAM-(pyridine)421 The procedure used to synthesize G2 which was also applied to synthesized G6 (183 mg, 79%). 1HNMR (400 MHz, D2O) : 8.60 (d, J = 5.6 Hz, 421 H), 8.41 (t, J = 8 Hz, 421 H), 7.93 (d, J = 8 Hz, 421H), 7.83 (t, J = 6.4 Hz, 421H), 4.18 (s, 842 H), 3.48~3.25 (m, 2648 H), 2.75~2.66 (m, 1420 H). Mass (MALDI, m/z) calculated M: 1439.5 (M + Na+ + 66H+ )/67, 3027.2 (M + 14Na+ + 5K+ + 13H+ )/32, 1347.4 (M + 12Na+ + 9K+ + 51H+ )/72; Found: 1439.5; 3027.2; 1347.5

2.2.6 Characterization of the number of dendrimer-bonded pyridine groups The number of introduced pyridines was first determined by NMR, comparing the integral of the aromatic region with that of the aliphatic region. The obtained pyridine number was further confirmed by MS spectra. The results indicated that this reaction could functionalize from 82% to 85% of the surface amino groups of all generations except G2, which contained 90% of surface group. Several attempts, including a prolonged reaction time, and the increase of the equivalents of pyridine aldehydes, failed to improve the functionalization number of peripheral pyridines. Presumably, the steric hindrance of the pyridine surface groups prevented a greater functionalization. 7 ACS Paragon Plus Environment


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2.3 EPR sample preparation Solutions containing Cu(NO3)2·3H2O at concentrations from 0.0025 M to 0.1 M and the Gn dendrimers at concentrations from 0.01 M to 0.2 M in surface groups were prepared in TRIS-buffered saline (TBS – at pH = 7.9) water solvent. The concentrations could not be lower than the minima reported above, due to the sensitivity limits of the technique. The blanks constituted by Cu(NO3)2·3H2O solutions in the same solvent at concentrations from 0.0025 M to 0.1 M were analyzed as references. Other solutions were prepared in the same solvent containing DTT at DTT/Cu(II) molar ratios from 0.1 to 2, or AA at AA/Cu(II) molar ratios from 0.1 to 10. In all solutions, the spin-trap PBN was added at a concentration of 5 mM. The Cu(II)/dendrimer-surface groups molar ratio was changed holding constant the concentration of the dendrimer or, alternatively, that of Cu(II). The samples were left equilibrating from 1 to 24 h showing that the spectra did not change in this time range.

2.4 EPR Instrumentation and Method

EPR spectra were recorded by means of an EMX-Bruker spectrometer operating at X band (9.5 GHz) and interfaced with a PC (software from Bruker for handling the EPR spectra). The temperature was controlled with a Bruker ST3000 variable-temperature assembly cooled with liquid nitrogen. The EPR tubes have a constant internal diameter of 2 mm and were filled with 50 µL of solution. The setup of the instrument was the same for all the EPR measurements. The EPR spectra were recorded for the different samples as a function of temperature, but the spectra were only computed at 298 K and 150 K. In all cases, we controlled the reproducibility of the results by repeating the EPR analysis (three times) in the same experimental conditions for each sample. The spectra reproducibility in the same 8 ACS Paragon Plus Environment

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experimental conditions allowed us to evaluate the absolute intensity of the EPR spectra, measured as double integration of the derivative EPR signals.

2.5 EPR spectra computation

Computation of the EPR spectra was performed by using the well-established procedure of Budil and Freed [24], which allowed us to obtain structural and dynamical information on the systems. The main parameters extracted from computation were: (i) the gii components for the coupling between the magnetic field and the electron spin (accuracy ± 0.001). As internal reference having = 2.0036, 1,1diphenyl-2-picryl-hydrazyl (DPPH) standard was added to the solutions. The accuracy in the gii parameters is ± 0.0001; (ii) the Aii components for the coupling between the electron spin and the Cu(II) nuclear spin (I = 3/2). The accuracy in these parameters estimation is ± 0.5 G; (iii) the line width, indicated as LW (accuracy ± 0.5 G); and (iv) for the spectra at 298 K, the correlation time for the rotational diffusion motion of the Cu(II) ions and their complexes, indicated with τ (accuracy ± 0.01 ns). In several cases, the spectra were constituted by different signals, i.e., different spectral components which corresponded to Cu(II) in different coordination sites. A subtraction procedure from one to another experimental spectrum allowed us to extract the different components, to quantify them by means of a double integration of the signals, to evaluate the relative percentages (accuracy ± 1%), and to separately compute the different components to obtain the structural and dynamical information. The gii and Aii magnetic parameters extracted from the simulation were then compared with the parameters previously obtained for Cu(II)-dendrimer complexes [18-24]. This allowed us to assign each spectral component to a copper coordination and identify the structure and complexing sites of the dendrimers.



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3 Results and Discussion

3.1 Cu(II)-dendrimer complexation in the absence of the reducing agents

The Cu(II)-dendrimer complexation was analyzed by EPR in the absence of the reducing agents to be compared to complexation in the presence of the reducing agents. Table 1 shows the number of reactive Cu(II) ions determined from spectrophotometric titration [17]. Also, the number of Py groups is listed for each dendrimer.

Table 1 Number of reactive Cu2+ ions (Nact) and peripheral pyridine groups.

Nact

G2

G3

G4

G5

G6

4.8±0.7

7.6±1.4

22.2±3.9

57.3±1.2

81.7±0.7

28

53

109

218

421

n. of Py groups

EPR spectra were analyzed at both 298 K and 150 K for G2-G6 dendrimers in buffered (TBS) solutions at pH= 7.9 with increasing amounts of Cu(II). The dendrimer concentration (in surface sites) was also changed, but the spectra were comparable at the same Cu(II)/dendrimer molar ratio. For this reason,


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only the results obtained with a dendrimer concentration of 0.1 M in surface sites are henceforth presented. First, it is interesting to analyze the variation of the absolute intensity of the EPR spectra for the different samples as a function of the Cu(II) concentration (Fig. 2). As a reference, the variation of intensity for the Cu(II) solutions in the absence of the dendrimers (Blank) is also reported

Figure 2 Variation of the absolute intensity of the spectra of the Gn-Cu(II) solutions at 298 K as a function of Cu(II) concentration. Gn concentration is 0.1 M in Py groups. As a reference, the variation of intensity for the Cu(II) solutions in the absence of the dendrimers (Blank) is also reported

. As expected, the Blank showed a linear variation of the intensity as a function of the ion concentration. Conversely, by adding the dendrimers, the intensity decreased. For G2, already at the lowest Cu(II) concentration, corresponding to a 1:10 molar ratio between Cu(II) and the Py units, the intensity showed a decrease. The small size and the flexible structure of the G2 11 ACS Paragon Plus Environment


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dendrimer may be responsible of the intensity decrease. Considering a preferential binding of Cu(II) with the Py groups of the dendrimer, since the number of external Py groups of G2 is 28, at a 1:10=Cu(II):Py molar ratio, about 3 Cu(II) ions were binding to the dendrimer. This corresponded to more than 50 % of the total number of reactive Cu (II) interacting with G2. Further ions into such a small structure led to increasingly strong spin-spin interactions which provoked the quenching of the paramagnetism. Therefore, the intensity further deviated from the Blank with the increase in Cu(II) concentration. But, when the saturation of the interacting ability of the dendrimers was achieved, the ions were no more hosted by the dendrimer, and were confined externally to the dendrimer in the water solution. As a consequence, after saturation, the discrepancy between the intensity of the Blank and that of G2 samples remained almost constant. In agreement with this interpretation, the effect of spin-spin interactions diminished with the increase of dendrimer size from G2 to G4. In case of G4, the discrepancy in intensity between the Blank and the dendrimer was very small because the complexed ions were far from each other, mainly due to well distributed Py groups at the large external surface of the dendrimer available for Cu(II) complexation. But, for G5, the behavior was different: at the smallest Cu(II) contents, the intensity was similar to that of the blank, but, when saturation of well-geometricallydistributed Py groups was achieved, the intensity started decreasing with respect to the Blank and became similar to that of G2. This means that further ions approached the complexed ones and formed dimeric Cu(II)-Cu(II) species, which were EPR silent. The remaining EPR-visible ions for G5 were in similar amount as for G2. The situation for G6 was different since formation of dimeric Cu(II)-Cu(II) species occurred at a lower extent, probably due to the too high density of Py groups at the surface. To better understand the complexation behavior of these dendrimers with Cu(II), it is necessary to analyze the spectral line shape. Figure 3 shows some selected experimental spectra (normalized in height) of G4 dendrimers (0.1 M in surface groups) with increasing concentrations of Cu(II) at 298 K (a) and 150 K (b). 12 ACS Paragon Plus Environment

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G4 was selected as example since the signal-to-noise ratio was higher than for the other generations, and, consequently, the spectral variation as a function of Cu(II) concentration was better visualized if compared to the other generations. As indicated with arrows in Figs. 3(a) and 3(b), the spectra were constituted by two signals, which changed in their relative intensities by changing the Cu(II) concentration. (a)

(b)

(c)

(d)

Figure 3 Selected experimental EPR spectra (normalized in height) of G4 dendrimers (0.1 M in surface groups) with Cu(II) at increasing concentrations: 298 K (a); 150 K (b). Computations of the dendrimercomplex signals for a Cu(II) concentration of 0.0025 M at 298 K (c) and 150 K (b). 13 ACS Paragon Plus Environment


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Mainly, at the lower Cu(II) concentrations (≤ 0.03 M), only one component was present arising from dendrimer-complexed ions. Therefore, this component was called “dendrimer-complex signal”. An example of computation of this component for a Cu(II) concentration of 0.0025 M, performed using the program of Budil et al. [24], is shown in Fig. 3 for the spectra at 298K (c) and 150 K (d). The main parameters used for the simulation of the spectra at both low and room temperatures were: gii=2.036, 2.080, 2.239; Aii=15 G, 10 G, 175 G. For the spectrum at 298 K, =0.8 ns was also obtained from calculation. These parameters were characteristic of Cu(II) binding with four dendrimer nitrogen sites (Cu-N4 coordination) in a slightly rhombical-distorted square planar geometry and with a quite slow motion conditions [18-24]. These nitrogen sites could be the Py groups, but also internal tertiary nitrogen groups. Indeed, from previous studies on PAMAM dendrimers [18-20, 22-23], it is expected that also internal nitrogen sites are complexed. But, in a previous study it has been found that nitrogencontaining heterocycle groups are preferentially complexed [22]. As a consequence of this complexation, the dendrimer external surface may gain a positive charge which impedes the internalization of the ions. As a proof of this hypothesis, by increasing the Cu(II) concentration, saturation of the Cu-N4 coordination was achieved at a Py:Cu(II) molar ratio of about 4, and further ions were confined outside the dendrimers, interacting with a lower number of Py groups, mainly two Py groups and water (Cu-N2O2), in equilibrium with a Cu-O4 coordination (only water). The Cu-O4 signal obtained after saturation of the dendrimer-Cu(II) complexation was called “water signal”, as indicated with arrows in Figs. 3(a) and 3(b). When both the water and the dendrimer-complex signals were contributing (superimposing) to the spectra, the two spectral components were extracted and calculated as described in the Experimental section, to finally obtain the relative percentages of the two components.


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Figure 4 shows the variation of the relative percentage of the dendrimer-complex signal as a function of the Cu (II) concentration (at a constant concentration of the dendrimers of 0.1 M in Py groups) for the various dendrimers.

Figure 4 Variation of the relative percentage of the dendrimer-complex signal as a function of the Cu (II) concentration for the dendrimers from G2 to G6 at a concentration of 0.1 M in surface groups. It resulted that, for the dendrimers from G2 to G5, the water signal was not recordable (100 % of the Dendrimer-Complex signal) up to the Cu-N4 saturation condition corresponding to [Cu2+] = 0.03 M, that is, about a 1/4 molar ratio between Cu(II) and Py groups. However, above this ratio, the decrease in the relative dendrimer-complex signal percentage was different from one to another generation. For G2, the decrease was absent up to a 1:2 (Cu(II):Py) molar ratio, in agreement with formation of a preferential Cu-N2O2 coordination and an intensity decrease (Fig. 2), due to the small size and flexible structure of this dendrimer (favoring spin-spin interactions). The flexible structure favored the stabilization of the dendrimer-complexes, postponing the formation of the water complex. This effect 15 ACS Paragon Plus Environment


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decreased with the increase in generation from G2 to G4. In this last case (G4), the percentage of the dendrimer-complex signal drops down above the Cu-N4 saturation conditions since all the ions are contributing to this complex (high intensity in Fig. 2). At Cu(II)/Py molar rations > 1:4, the ions were repulsed outside the dendrimer, coordinating less than four Py groups (mostly 2) and water molecules, and then only sitting in the water solution. Conversely, G5 did not follow the trend: up to a Cu(II)/Py molar ratio = 0.5 it behaved similarly to G4, since the amount of dimeric Cu(II)-Cu(II) complexed species was equivalent to the discrepancy in Py number between G4 and G5; but, at the highest Cu(II) concentrations, it showed a dendrimer-complex relative percentage much higher than expected from the G2-G4 trend, even similar to G2. This is because, after saturation of Cu-N4 complexation, all the excess of Cu(II) was used for the formation of dimeric complexes, which were favored by the dense structure of the G5 dendrimer at the external layer. These dimeric complexes are EPR silent in the investigated range of temperature, but we see that the water signal contributed with smaller relative percentage than expected from the trend from G2-G4 dendrimers. In G6 case, due to the too high density of Py groups, the Cu(II) complexation, also forming dimeric species, created a positively charged surface which repulsed the ions and favored their extrusion in the water solution. Therefore, the water signal for G6 appeared at a Cu(II)/Py molar ratio of about 0.2 and increased its relative percentage as a function of Cu(II) concentration more efficiently than the lower generations. So, on the basis of the EPR analysis, G5 dendrimer showed a peculiar interacting ability in respect to Cu(II) ions, due to the optimal distribution of Py groups at the external surface, which well justifies the highest value of Vmax found in previous studies [17]. Further information came from the magnetic parameters obtained by calculating the spectra. For the dendrimer-complex signal, the computations shown in Figs. 3(c) and (d) were obtained for a Cu(II) concentration of 0.0025 M and the spectra were quite similar for all generations, characteristic of a CuPy4 coordination with a square planar structure, which was increasingly distorted by increasing 16 ACS Paragon Plus Environment

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generation. This distortion, arising from an increased density of Py groups at the dendrimer surface, led to a small increase in the gii parameters and a decrease in the Aii parameters. Among these parameters, the most reliable to follow the spectral variations was the gxx value, which was obtained from spectral computation assuming a constant Axx value. Figure 5 shows the variation of gxx in the dendrimercomplex signals for the various dendrimers at 150 K as a function of Cu(II) concentration.

Figure 5 Variation of gxx in the spectra for the various dendrimers (0.1 M in surface groups) at 150 K as a function of Cu(II) concentration. The gxx value increased with the increase of Cu(II) concentration, but a gxx increase may arise from different sources: (i) a distortion of the square planar structure of the Cu-Py4 complex, which was already indicated as responsible of the variations from one to another generation at 0.0025 M. However, this distortion cannot justify the large gxx variation obtained by increasing Cu(II) concentration; (ii) the weakening of the Cu(II)-N binding strength. This effect may explain only small variations of gxx, like those progressively transforming one coordination to another one; and (iii) the formation of different Cu(II)-dendrimer complexes with a lower amount of bonded Py groups. Specifically, the gxx ≤ gyy < gzz 17 ACS Paragon Plus Environment


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sequence was preserved for all spectra and this indicated that the square planar structure was maintained, with some distortions. But, a lower number of nitrogen ligands (Cu complexing from 4 to 0 Py groups, and from 0 to 4 water molecules) produces a decrease of the gii parameters as large as found in Fig. 5 and was therefore responsible of the variation of gxx by increasing Cu(II) concentration. However, it is interesting that the variation of gxx was different for different generations. G2 showed an almost negligible variation of gxx up to [Cu2+] = 0.02 M, while the higher generations showed variations since Cu(II) concentrations of 0.005 M. This means that, for the higher generations, a small amount of Cu(II) was enough for creating distorted complex structures, due to a decreased flexibility of the dendrimer branches and a weakening of the Cu(II)-N binding. Very probably, at the highest Cu(II) concentrations, strong spin-spin interactions and charge repulsion between Cu(II) binding in close sites created a distribution of complexes with different Cu(II)-N binding strengths and formation of further coordination with 1-2-3 pyridine groups. On the basis of the magnetic parameters, the Cu-N2O2 coordination was the prevalent one, in line with the presence of adjacent Py groups, but Fig. 1 also shows that some dendrimer branches are functionalized by a single Py group, and this justifies further coordination with 3 or only 1 Py group (Cu-N3O and Cu-NO3, respectively). G4 and G6 showed the highest gxx values, and behaved in a similar way by increasing Cu(II) concentration, but for different reasons: in G4 all the ions were contributing to the EPR spectra with the progressive formation of different dendrimer complexes, involving different amounts of Py ligands from 4 to 1; for G6 the high density of surface groups impeded the formation of geometrically well-structured complexes and easily saturated the dendrimer complexation ability. Finally, for G5, a peculiar behavior was found, since a fraction of ions gave rise to formation of dimeric species which were EPR silent, while the remaining ions showed a complexation behavior similar to that of G3, by forming stable Cu(II)-Py complexes, mainly coordinating 2 Py groups in the Cu(II)/Py molar-ratio range between 0.3 and 0.5. 18 ACS Paragon Plus Environment

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3.2 Cu(II)-dendrimer complexation in the presence of reducing agents

The Cu(II) -dendrimer complexation was analyzed in the presence of DTT and AA at increasing DTT/Cu(II) molar ratios from 0.1 to 2, and AA/Cu(II) molar ratios from 0.1 to 10. A much higher amount of AA with respect to DTT was needed to get a comparable reduction effect. DTT is known to bind Cu(I) tightly and caused the removal of Cu(I) ions from Py coordination sites [25]. In comparison, AA has only a mild effect on the same aspect. For this reason, the results about the effect of AA were reported in the Supporting Information (SI) and only shortly discussed in the following. We mainly focused the analysis on the results at the Cu(II) concentration of 0.05 M since it provided interesting information. Indeed, at this Cu(II) concentration, the EPR spectrum was characteristic of dendrimer-complexes formed at the external dendrimer surface, quite exposed to the attack of the reducing agents, mainly binding with 2 or 1 Py groups. A fraction of completely hydrated ions was also present. Higher or lower Cu(II) concentrations showed less informative results because there was the prevalence of the water signal or the Cu-Py4 dendrimer-complex signal, respectively. This latter signal, prevalent at concentrations equal or below 0.03 M in Cu(II), decreased in intensity in similar way for all generations (Fig. S1 in the Supporting Information (SI)). However, the variations of the parameters for each signal were comparable in respect to all Cu(II) concentrations at which that signal was present. Figure 6 shows the variations of the total spectral intensity (a) and of the intensity of the dendrimercomplex signal (b) for DTT as a function of the molar ratio between the reducing agent and Cu(II), at a Cu(II) concentration of 0.05 M. Equivalent graphs are reported in the SI (Fig. S2) for AA (Fig. S2(a) and S2(b)), for the total and relative to the dendrimer complex intensities, respectively), at a Cu(II) concentration of 0.05 M. Before analyzing these data, it is important to underline that the intensity of the dendrimer-complex signal is considered in Fig. 6 instead of the relative percentage of this signal used for Fig. 4, because, in the presence of reducing agents, it resulted informative about the reducing 19 ACS Paragon Plus Environment


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effect to compare the total and relative intensities, instead of the percentages of Cu(II) bonded or unbonded to the dendrimer. About DTT effect, for all generations the intensity went to zero at the 1:1 molar ratio between Cu(II) and DTT, but the intensity decrease was more efficient since the smallest DTT concentrations for G3 and G4 with respect to the other generations. On the basis of the results in the absence of the reducing agents, G3 and G4 dendrimers showed a better distribution of Cu(II) ions at the dendrimer surface, thus avoiding strong spin-spin interactions which were responsible of the intensity decrease in Fig. 2. Instead, G2, G5 and G6 suffered of strong spin-spin interactions for different reasons: G2 due to the small size and the flexible structure, while G5 and G6 due to the high density of surface groups and the formation of Cu(II)-Cu(II) dimeric species. As already underlined, strongly spin-spin interacting ions were “EPR silent”. The results in Figs. 6(a) and 6(b) indicate that DTT had to reduce also the EPR-silent ions sitting in close position for G2, G5 and G6 samples, and, therefore, in these cases the spectral intensity was changing less than expected. (a)

(b)

Figure 6 Variations of the total spectral intensity (a) and of the intensity of the dendrimer-complex signal (b) as a function of the molar ratio between the reducing agent and Cu(II), for DTT at a Cu(II) concentration of 0.05 M. 20 ACS Paragon Plus Environment

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This behavior was similar for the total intensity and the intensity of the dendrimer-complex signal, even if, at the lowest DTT/Cu(II) molar ratios, Cu(II) complexed by G2 showed a more efficient intensity reduction of the dendrimer-complex signal if compared to G5 and G6, due to a negligible presence of the water signal for G2 at 0.05 M of Cu(II) (Fig. 4). But, interestingly, from a DTT/Cu(II) molar ratio above 0.4, the dendrimer-complex signal was equivalently and apparently-slowly reduced for both G2 and G5. Again the reason is the small size and open-flexible structure for G2, while G5 had a high concentration of EPR-silent dimeric-copper complexes to be reduced by DTT. For AA, as shown in Fig. S2 (SI), even at a AA/Cu(II) molar ratio = 10, the total intensity was less than halved in all cases even if the intensity decrease occurred at AA/Cu(II) molar ratios up to 1. This is a further proof that the Cu(I) ions that originated from the reduction reaction remained at the dendrimer surface and impeded to the further AA molecules to approach the Cu(II) ions bonded into the dendrimer surface. Conversely, DTT efficiently removed the Cu(I) ions by complexing them [25]. A comparison between the variations of the total intensity and the intensity of the dendrimer-complex signal in Fig S2 showed that the lowest amounts of AA were almost completely dedicated to reduce Cu(II) at the dendrimer-water interface, but then, amounts of AA larger than 1 molar ratios were largely used to partially reduce Cu(II) ions more strongly coordinated to the dendrimers. This mainly held for the smaller generations (G2-G4), since the higher generations, mainly G5, offered the dimeric complexes to the reducing agent and the intensity could not diminish as much as expected. Further information was obtained by evaluating the parameter Hmin indicated in Fig. 3(a) for the room temperature spectra. This empirical parameter well resembles, in respect to its variations, the calculated ones obtained as = (Axx+Ayy+Azz)/3 and , and provides simple and direct information on the coordination behavior and flexibility of Cu(II) when interacting with the dendrimers. In a better detail, and consequently Hmin increase with the increase in the number and strength of Cu(II)-coordinated 21 ACS Paragon Plus Environment


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nitrogen sites. In a different point of view, if the rotational mobility of the Cu(II)-dendrimer complex slows down (increase in ) due to a lower flexibility of the Cu(II)-dendrimer complex, the magnetic parameters become anisotropic and Hmin increases, that is, moves to higher magnetic field values. Figure 7 shows the variations of Hmin as a function of DTT/Cu(II) molar ratios for the various dendrimers (the plot for the variation of Hmin as a function of AA/Cu(II) molar ratio is reported in Fig. S3 - SI). The Cu(II) concentration is 0.05 M and the dendrimer concentration is 0.1 M in Py groups. Hmin increased as a function of the reducing-agent concentration. This indicated that the reduction occurred first on the Cu(II) ions coordinating with less than 4 Py groups at the dendrimer external surface. This is expected since these ions are more exposed to the reducing agent attack. Conversely, the Cu-Py4 complexes are inside the dendrimer external surface, and, therefore, quite protected and less mobile. So, progressively, in the DTT case, the coordination Cu-Py, Cu-Py2 and Cu-Py3 became EPR invisible and finally, also the ions coordinating with 4 Py groups were reduced.

Figure 7 Variation of Hmin as a function of DTT/Cu(II) molar ratio for the various dendrimers. The Cu(II) concentration is 0.05 M and the dendrimer concentration is 0.1 M in Py groups.


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But, as already found for the intensity variation (Fig. S2), the Hmin variations for AA (Fig. S3) indicated a weaker reducing effect if compared to DTT, being the reducing action of AA more effective on G2, while G6 showed the smallest variation of Hmin due to the too congested surface groups and the consequent prevalence of water complexes.

3.3 Spin-trapping experiments and detection of ROS

In all experiments in the presence of the reducing agents, the spin trap PBN was added to the system in order to detect by EPR the eventual formation of transient radicals, mainly reactive oxygen species (ROS). A very low intensity radical signal was clearly visible in the EPR spectra, mainly for DTTcontaining samples, starting from equimolar DTT and Cu(II) amounts, at Cu(II)/Py molar ratios in between 0.3 and 0.5, for all generations, mainly G2 and G5, but excluded G6. The rationale of these outcomes was explained on the basis of all the results described above, as follows: (i) equimolar DTT and Cu(II) amounts led to the complete reduction of Cu(II). This both increased the probability to form ROS from the reduction reaction, and cleaned the spectrum from the Cu(II) signals; (ii) G6 showed the prevalence of the water complex, since the ions were mostly embedded into the packed dendrimer surface. In these conditions the spin trap was probably poorly accessible and, also, radical quenching occurred; and (iii) G2 and G5 showed strong spin-spin interactions between Cu(II) externally bounded at the dendrimer surface (mainly Cu-Py2 complexes). Therefore, these dendrimers showed a higher local concentration of Cu(II) ions ready to be reduced and locally forming the radical species. Mainly G5, forming stable dimeric complexes at the surface of big, slow moving nano-molecules, offered a spacefixed region of ions where the reduction process and the ROS formation easily occurred and the radical might stabilize.



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An example of PBN-trapped transient radical for G2 dendrimer with Cu(II) and DTT concentration of 0.03 M, is shown in Fig. 8. Isotropic nitrogen and proton hyperfine coupling constants were obtained from computation. It was found: = 16.5 G and = 3.7 G.

Figure 8 PBN-trapped transient hydroxyl radical for G2 dendrimer (0.1 M) with Cu(II) and DTT at concentration of 0.03 M. PBN added at 5 mM concentration. Computation is shown as red line. A comparison with similar values found in the literature for the same spin trap in water solutions allowed us to identify this radical with the hydroxyl (.OH) one [26-27]. We cannot exclude that different, but shorter living, radicals were formed too. Indeed, the hydroxyl radical may arise from degradation of superoxide anion radicals, which are too short living in such fluid environment. The computation also provided the microviscosity parameter:  = 0.05 ns, which indicates that this radical was captured by the spin trap in the water interphase at the dendrimer surface.


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4 Conclusions

Pyridine decorated PAMAM dendrimers from generation 2 to generation 6 were synthesized and their complexation behaviour with Cu(II) at different molar ratios between the ions and the dendrimer surface sites was analysed by using EPR in the absence and presence of the reducing agents DTT and AA, and the PBN spin trap. At Cu(II)/Py molar ratios equal or below 0.3, Cu(II) was only forming a Cu-Py4 complex in a slightly rhombical-distorted square planar geometry and with a middle-slow motion conditions. The distortion increased by increasing generation due to increased Py congestion at the dendrimer surface. By increasing the Cu(II)/Py molar ratio the coordination progressively changed showing a weakening of Cu-N bonds and the substitution of Py groups with water molecules. The complexes from Cu-Py3 to Cu-Py, but preferentially Cu-Py2, progressively stabilized at the dendrimer external surface in structures more exposed to the solution, and therefore Cu(II) was easier reduced by the reducing agents, mainly DTT which formed stable complexes with Cu(I). For all generations, the intensity went to zero at the 1:1 molar ratio between Cu(II) and DTT. Therefore, the most external ions at the dendrimer surface, complexed with water, 1 Py and, mainly, 2 Py groups were first reduced by DTT and AA. After this, DTT was able to continue the reducing action on the Cu(II) ions complexed with 3 and 4 Py groups. By using PBN spin trap, hydroxyl radicals were identified at the dendrimer surface. They may arise from degradation of superoxide anion radicals, which are too short living in such fluid environment. In respect to the behavior of the different generations, the main information may be resumed as follows. For G2, when more than 50 % of the total number of reactive Cu (II) were interacting with G2, further ions into the small-dendrimer structure led to increasingly-strong spin-spin interactions. By increasing the Cu(II)/Py molar ratio, the Cu-Py4 complex transformed into a Cu-Py2 one without forming the water



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complex up to a Cu(II)/Py molar ratio of 0.5. This dendrimer showed a higher local concentration of Cu(II) ions ready to be reduced by DTT and AA, and locally forming the radical species. For G4 (G3 showed an intermediate behavior between G2 and G4 in the Cu(II) complexation), the complexed ions were well distributed at the large external surface of the dendrimer, thus avoiding spinspin interactions. By increasing the Cu(II)/Py molar ratio, all ions contributed to the EPR spectra, and, therefore, the preferential Cu-Py2 complexation stabilized at a Cu(II)/Py molar ratio of 0.5. However, at this molar ratio, already 45 % of ions were forming the water complex, probably due to a charge repulsion effect. By adding DTT, the intensity decrease was more efficient for G3 and G4 with respect to the other generations, already at the smallest DTT concentrations due to stable and well-balanced complex distribution at the dendrimer surface. However, the spin trapping process of the hydroxyl radical was less efficient for G3 and G4 than for G2 and G5. For G5, when saturation of well-geometrically-distributed Py groups was achieved, further ions approached the complexed ones and formed dimeric Cu(II)-Cu(II) species. This effect avoided an increase in the relative percentage of water complex with respect to G4, and, at Cu(II)/Py molar ratio equal to 0.5, the percentage of water-complexed ions was still 45 % and poorly increased by increasing Cu(II) concentration. Therefore, a peculiar behavior was found for G5, since a fraction of ions gave rise to dimeric species which were EPR silent, while the remaining ions showed a complexation behavior similar to that of G2-G3, by forming stable Cu(II)-Py complexes, mainly coordinating 2 Py groups in the Cu(II)/Py molar ratio between 0.3 and 0.5. This peculiar complexation behavior, due to an optimal distribution of Py groups at the external surface, justifies the catalytic ability described in previous studies [17]. G5, forming stable dimeric complexes at the surface of big, slow-moving nano-molecules, offered space-fixed ions available to the reducing agents. Consequently, the ROS formation easily occurred and the radicals stabilized.


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For G6, on the basis of the EPR results, formation of dimeric Cu(II)-Cu(II) species occurred at a lower extent, probably due to a too high density of Py groups at the surface. In this case, the Cu(II) complexation created a positively charged surface which repulsed the ions and favored their extrusion in the water solution. For this reason, the water complex already formed at a Cu(II)/Py molar ratio of 0.2 in equilibrium with the Cu-Py4 complex. Therefore, for G6, the reduction mainly involved the water complex due to the too congested surface groups and the consequent extrusion of the ions to the interphase. No radicals were evidenced after completing the reduction process at the G6 surface. This study provided information on the complexation behavior of the PAMAM-Py dendrimers at different generations in respect to increasing amounts of Cu(II), which may be used for biomedical purposes. But, mainly, it demonstrated that the EPR technique was very useful to give insights on the reduction process of the Cu(II)-dendrimer complexes and the eventual formation of ROS, involved in biocatalysis.

Acknowledgements

MFO and MC thank the master student Silvia Marchionni for her contribution in sample preparation and EPR analysis. We are also grateful to the Department DiSPeA, University of Urbino, for the financial support. YHT and CLK thank MoST, Taiwan for their financial support.

Supporting Information: Supplementary material: Fig. S1: Variation of the total spectral intensity, equal to the intensity of the dendrimer-complex signal (Cu-Py4) as a function of the molar ratio between DTT and Cu(II), at a Cu(II) concentration of 0.01 M for the various dendrimers (0.1 M in surface groups); Fig. S2: Variation of the total spectral intensity (a) and of the intensity of the dendrimercomplex signal (b) for AA as a function of the molar ratio between the reducing agent and Cu(II), at a 27 ACS Paragon Plus Environment


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Cu(II) concentration of 0.05 M; Fig. S3: Variation of Hmin as a function of AA/Cu(II) molar ratio for the various dendrimers. The Cu(II) concentration is 0.05 M and the dendrimer concentration is 0.1 M in Py groups.

References

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EPR Characterization of Copper(II) Complexes of ... - ACS Publications

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