Gold Nanoparticles Modified with Guanine and Its Derivatives: Study

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Gold Nanoparticles Modified with Guanine and Its Derivatives: Study of Conformational Changes Svetlana Avvakumova,*,† Paolo Verderio,‡ Giovanna Speranza,† and Francesca Porta*,† †

Dipartimento di Chimica, University of Milan, via Golgi 19, Milan, Italy Dipartimento di Biotecnologie e Bioscienze, University of Milan “Bicocca”, Piazza della Scienza, 2, Milan, Italy

‡

S Supporting Information *

ABSTRACT: The interactions between DNA and AuNPs is one of the most interesting subjects of modern nanotechnology, giving rise to plenty of applications. The studies on the identification of the binding sites between DNA components and gold nanoparticle are continuously in progress; however, information about how the interaction influences the conformation of guanine compounds is still lacking. In this paper, we report on the preparation of AuNPs modified by guanine ligands via a one-pot reduction method in aqueous solution. The nanoparticles were fully characterized by UV−vis, TEM, DLS, and zeta-potential methods. The research on gold−ligand binding sites and the conformational changes in the molecular structure of ligands was performed making use of ATR-FTIR and NMR techniques. Notably, novel interesting results on the conformational rearrangements of ribose moiety were obtained.

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INTRODUCTION

by Mirkin et al., using absorption of purines and pyrimidines from solution on flat gold surfaces.17,18 The chemistry of nucleosides and nucleotides in solution and solid phase is well-known, mainly by spectroscopic methods, such as X-ray diffraction, NMR, and FTIR.19,20 In particular, in NMR spectroscopy chemical shifts and coupling constants help to investigate possible sugar conformations,21 while in the IR spectra the ribose puckering can be derived by the position of ring breathing band(s).22 To date the interaction of purines with gold nanoparticle surface and the stability of nonthiolated systems have not been fully studied. A recent article highlights the status of art on the literature in this field and describes the behavior of nucleosides adsorbed on a metallic surface of Au(100) using density functional theory computations.23 With our experimental research addressing a basic knowledge of chemical and structural properties of organic−inorganic hybrid systems, we decided to investigate the chemistry of guanine compounds as ligands for colloidal gold nanoparticles, because of the good reproducibility of the systems. Herein, we report on a facile and universal route to synthesize watersoluble small AuNPs (6−16 nm), using a set of capping ligands as stabilizers: inosine (Ino, 1), guanine (Gua, 2), guanosine (Guo, 3), guanosine 5′-monophosphate (5′-GMP, 4), and some derivatives, i.e., 1-methylguanosine (M1G, 5) and 8bromoguanosine (8-Br-Guo, 6). The chemical structures of 1− 6 molecules lead us to believe they could be effective capping ligands for AuNPs: 1 and 2 offer covalent interacting lone pairs,

In the past decades, gold nanoparticles (AuNPs) have attracted much attention in many fields of biology and medicine, such as drug delivery, cellular imaging, and biological and chemical sensing.1−4 AuNPs provide a particularly useful tool, demonstrating unique properties with potentially wide-ranging therapeutic applications, with their dimensions being comparable to the size of protein molecules. In particular low toxicity and stability under physiological conditions are required in their biological applications.5−8 The uptake of purines in cancer cells has been studied elsewhere.9,10 It was reported that driving cells toward differentiation pathways could be a novel therapeutic approach, since cancer is regarded as a disease of cell maturation rather than cell multiplication.9 Therefore, the realization of treatments capable of activating normal pathways of differentiation in malignant cells can be of interest. In this concern, purine nucleotides, due to their pharmacological function and nontoxic nature in normal cells, are regarded as new therapeutic agents against various pharmacological events including various types of cancers.10 Among purines, guanine-based nucleotides are important on account of their ubiquitous role signaling molecules in extracellular compartment, aside from their routine intracellular functions. Moreover, chemical literature reports a growing number of studies on interaction between DNA and AuNPs as one of the most interesting subjects of modern nanotechnology, giving rise to plenty of applications, such as DNA chips and sensors, drug−DNA delivery, imaging, biodiagnostics, and electronics.11−16 Interesting studies on the identification of binding sites between Au and DNA components have been performed © 2013 American Chemical Society

Received: July 13, 2012 Revised: January 3, 2013 Published: January 4, 2013 3002

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Figure 1. Chemical structures of (1) inosine, (2) guanine, (3) guanosine, (4) guanosine 5′-monophosphate, (5) 1-methylguanosine, and (6) 8bromoguanosine.

reported in ref 23. To prepare AuNPs, to 5 mL of Milli-Q water was added an aqueous solution of NaAuCl4 (2 × 10−3 mmol; 34.1 mM). Under vigorous stirring, an aqueous solution of 1methylguanosine (5) (1 × 10−5 mmol, 0.272 mM), 8bromoguanosine (6) (2 × 10−5 mmol, 0.263 mM), or inosine (1) (1 × 10−5 mmol, 0.69 mM) was added. After 5 min, an aqueous solution of NaBH4 was added (4 μmol; 0.1M), and red sols were immediately formed. The obtained colloidal solutions were left under stirring for several hours to complete the reaction. The particles were purified by dialysis overnight. We measured pH values during the reaction progress: the pH of the starting solution, before adding sodium borohydride, was quite acidic (pH 2.5−2.7), while the pH of the formed sols was close to 3.5−3.7 (likely due to traces of HAuCl4 present in the NaAuCl4 solution). Gold Nanoparticle Characterization. UV−vis spectroscopy was used to follow the reaction progress and estimate nanoparticle stability, while dynamic light scattering and zetapotential measurements were carried out to evaluate the particle electrokinetic characteristics. The nanoparticle morphology was studied by TEM microscopy. The ligand shell was characterized by ATR-FTIR and 1H and 31P NMR spectroscopy (see Supporting Information for further details). Because of the high ligand concentration required for FTIR and NMR studies, the samples for these measurements were prepared by scaling up the quantities of each reagent. The coverage of nanoparticles (number of ligands per nanoparticle) was found both from theoretical calculations (see Supporting Information) and from quantitative analyses (ICP and UV−vis).

while 3, having a charged phosphate group, offers also electrostatic interaction.17 Guanine, one of the important building blocks of nucleic acids, has the maximum number of tautomers, the lowest ionization potential, and the maximum negative vertical electron affinity among all the nucleobases.25 To evaluate the influence of the substitution on purine ring in the coordination to gold, 5 and 6 derivatives are taken into consideration. Hence, the main objective of the present research is to discover which kind of interaction (covalent, polar, or hydrogen bond) occure between gold surface and guanine compounds, using the data coming from IR and NMR experiments.

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EXPERIMENTAL SECTION Synthesis of Gold Nanoparticles. The solutions were freshly prepared for each synthesis, except for sodium tetrachloroaurate(III), which was prepared as a 34.1 mM stock solution. With guanine being insoluble in water at room temperature, the solution was prepared dissolving it in 0.01 M NaOH, followed by dilution with water up to the necessary concentration. All the glass vessels and stirrers were washed with aqua regia (Caution! Extremely corrosive!), and the solutions were filtered through a 0.22 μm cellulose filter prior to use in order to avoid the undesirable nucleation. 1. Au−Guanine (16 nm), Au−Guanosine (16 nm), and Au−Guanosine 5′-Monophosphate (6 nm) NPs. To 5 mL of Milli-Q water was added an aqueous solution of NaAuCl4 (2 × 10−3 mmol; 34.1 mM). Under vigorous stirring, an aqueous solution of guanine (2) (8 × 10−6 mmol, 0.909 mM), guanosine (3) (1 × 10−5 mmol, 0.42 mM), or guanosine 5′monophosphate (4) (6 × 10−4 mmol, 1.18 mM) was added. After 5 min, an aqueous solution of NaBH4 was added (4 μmol; 0.1M), and red sols were immediately formed. The obtained colloidal solutions were left under stirring several hours to complete the reaction. The particles were purified by dialysis overnight (Sigma-Aldrich dialysis tubing cellulose membrane, cut off 12.400 MW). 2. Au−1-Methylguanosine (13 nm), Au−8-Bromoguanosine (13 nm), and Au−Inosine (10 nm) NPs. 1-Methylguanosine (5) and 8-bromoguanosine (6) were synthesized as

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RESULTS AND DISCUSSION Small water-soluble gold nanoparticles (AuNPs) were prepared by one-step reduction of a gold salt aqueous solution with sodium borohydride in the presence of (1−6) purine derivatives as capping ligands (Figure 1). The ligand/gold molar ratios were tuned in order to obtain long-term stable nanoparticles, while the reducing agent was used in excess with respect to Au (2:1 mol/mol) to guarantee the complete reduction (S1, Table 1).26 By UV−vis spectroscopy, we controlled AuNP formation and determined the reaction 3003

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appear as chainlike structures, likely formed under a natural dewetting process by slow evaporation of nanoparticlecontaining solvent on the TEM grid (Figure 2A,E,F).27,28 Dynamic light scattering studies confirmed TEM results, showing the presence of fractions with larger mean diameters (95 nm for Au-Gua and 64 nm for Au-Guo), arising from the formation of small particle aggregates (Table 1). In this study we used both UV−vis and zeta-potential techniques to estimate the colloidal stability. The zeta-potential values of AuNPs capped by purine derivates were found rather negative compared to +11.04 mV value for “bare” nanoparticles, prepared using all the reagents in the same molar amounts without adding any ligand (Table 1). Although the highly negative value of Au−5′-GMP NPs (−47.56 mV) can be ascribed to the presence of negatively charged phosphate groups, also the values of Au−Gua and Au−Guo NPs are fairly similar and close to −25 mV, suggesting an important electronic contribute of 1−6 to the surface charge balance. The electrokinetic characteristics of some nucleosides and nucleotides have been studied elsewhere.29,30 Brook et al. have discovered that, under given reaction conditions, the AuNPs capped by adenosine phosphates are stable and possess negative Zeta-potentials, while AuNPs functionalized by nucleosides (e.g., adenosine or inosine) undergo aggregation caused by surface charge loss.29 We studied the nanoparticle stability both against pH- and salt-induced aggregation. As an

Table 1. Surface Plasmon Resonance, Hydrodynamic Diameter (DLS), Mean Particle Diameter (TEM), and ZetaPotential Values Au−ligand Au−Gua Au−Guo Au−5′GMP Au−M1G Au−8-BrGuo Au−Ino

SPR, nm

diameter (DLS), nm

diameter (TEM), nm

zeta potential, mV

524 525 525

19 (95) 16 (64) 24

16 16 6

−25.55 −21.56 −47.56

519 519

23 27

13 13

−20.25 −16.78

519

28

10

−22.07

termination by following the absorbance value and the position of surface plasmon resonance peaks. UV−vis data and reaction conditions are reported in detail in S1 of Supporting Information. Characteristic SPRs of AuNPs with the wavelengths varying between 519 and 525 nm, depending on the ligand used, are shown in Figure S3.1, A−F. The reactions were generally terminated after 2 h, when the absorbance stopped growing. The mean particle diameters (6−16 nm) were obtained by TEM microscopy (Figure 2). The images of Au−(2−4) NPs show the presence of small aggregates of easily observable nanoparticles (Figure 2B−D), whereas Au−(1, 5, 6) NPs

Figure 2. TEM micrographs of AuNPs capped by (1−6) ligands. 3004

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Figure 3. (a) Stability against pH-induced aggregation of AuNPs modified by guanine compounds. The pH values were 1, 3, 5, 7, 9, 11, 13. (b) UV− vis spectra of Au−guanine NPs in mQ water and different pH solutions.

empirical measurement of the aggregation process, we used an aggregation parameter, which measures the variation of the integrated absorbace between 600 and 700 nm. The aggregation parameter AP is defined as follows: AP = (A − A0)/A0, where A is the integrated absorbance between 600 and 700 nm of the sample at a given moment and A0 is the integrated absorbance between 600 and 700 nm of the initial, fully dispersed nanoparticle solution.31 Figure 3 shows the pHinduced aggregation patterns of AuNPs modified by guanine compounds. All the AuNPs were found stable in acidic, neutral, and basic pH (from 1 to 11), showing the AP less than 0.1. On the other hand, the SPR band was found broadened moving to pH 13, indicating a slight aggregation of the particles (AP < 0.6). Au-5′-GMP NPs were found to be more stable than others, showing a lower AP value. As for salt-induced aggregation, as-prepared AuNPs were found to aggregate in high-salt-concentration solutions (1−3 M NaCl). Guanine has several tautomer forms, the stability of which is significantly influenced by the reaction medium. The reported IR studies reveal that both keto- and enol-guanine tautomers are present in equal proportions in solution, although the ketoN(9)-H form dominates in polar solvents.32 In addition, the presence of protonated forms of guanine compounds, generated in acidic solution, must be considered. In order to understand in which form (neutral or protonated) the guanine derivatives exist under our reaction conditions (aqueous solution at pH ∼ 3.5), we calculated the values of concentration fractions (α) as function of pH, using the acidity constant logarithms, pKa. From these, one can readily calculate an approximate ionization state of the species at any pH value in aqueous solution.33 The summary of pKa and α values at pH 3.5 is reported in Table S2 in Supporting Information. We can conclude that guanine, 2, exists in the protonated form at 40% (protonated at N(7) atom) at 3.5 pH, while 1, 3−6 compounds are not protonated (neutral). The protonation state of compounds 1−6 influences directly their spectroscopic characteristics and the way they interact with gold surface. Structural investigations of the ligand shell surrounding gold nanoparticles have been already performed by IR techniques to characterize the ligand−gold interactions. In particular, Mirkin and co-workers have used the temperature-programmed desorption technique to study the interaction of DNA bases and nucleosides with a gold thin film, deposited on a flat

support. They concluded, considering guanine, that 2 forms a dispersed monolayer phase on Au, at low coverage, in which Hbindings between neighboring molecules are unlikely to occur. Moreover, the observed strong CO stretching peak suggests that the guanine molecular plane is not parallel to the metal surface.17 Pergolese et al. have studied the adsorption of guanine, guanosine, and 2′-deoxyguanosine on gold colloidal particles by SERS spectroscopy, suggesting that the molecules are tilted respect to gold and the sugar moiety is not involved in the interaction.34 Finally, Kryachko et al. have found that guanine interacts with Au3,4 clusters via O(6)−N(7) or O(6)− N(1) atoms, throughout a nonconventional N−H···Au hydrogen bond.35 1 H and 31P NMR spectroscopies have been widely used to study nucleobases or other nucleic acid components coordinated to metal centers;36−38 thus, we used the coordinative metallorganic approach to explain the interaction of 1−6 compounds with gold surface, comparing IR and 1H NMR spectra of free ligands, with the related spectra of Au−(1−6) NPs.36,39 In theory, the metal core can interact with ligands in many ways: via phosphate oxygen atoms, sugar oxygen atoms, and (N, C, O) atoms of the heterocyclic base, or combinations thereof.36 It should be noted that guanine N(7) atom possesses a high basicity and a favorable electrostatic potential to be the target for the metal coordination. A large dipole moment of guanine and its orientation does allow the coordination of any positively charged metal entity to this site.36 Moreover, an inspection of the structures of guanine−gold complexes (see Supporting Information, Figure S3.2), as well as gold complexes with other aza-aromatic rings, invariably shows N−Au chemical bonds of average length of 0.21 nm. This indication seems to point to a preferred adsorption mode in which the molecular rim (N-atoms) points toward the metal. TPD-MS desorption energies for guanine on gold are 127−139 kJ mol−1,17 indicating a strong interaction and ruling out any adsorption−desorption equilibrium at room conditions. Calculated (by density functional theory) attachment energies for nucleosides on Au(100) are 30−70 kJ mol−1,23 significantly lower than the above-mentioned desorption energies. 1 H and 31P NMR Data. A comparison of 1H and 31P chemical shifts at pH 7 and 3.5 of (1−6) guanine compounds and Au−(1−6) nanoparticles is reported in Table 2 (an 3005

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with free neutral guanine (7.65 ppm, δ = 0.63 ppm) and even more than that of free guanine in acidic conditions (7.98 ppm, δ = 0.3 ppm) (Figures S4.3.1−S4.3.2, Table S4.1). The change must be attributed to the electron density variation of the ring system, as a result of the gold interaction with the N(7) lone pair of nonprotonated guanine. The effect appears stronger than the deshielding effect observed upon protonation, suggesting an interaction of the basic guanine with a partially positive site on Au particle (i.e., Au(+I) centers on gold surface). The protonated guanine species (present at 40% in solution) does not seem to interact with the gold particle, as we observed the presence of only one H-8 signal in the 1H NMR spectrum of Au−(2) NPs. In 1H NMR spectrum of Au−guanosine (3) NPs, H-8 proton is slightly upfield shifted (δ = 0.06 ppm, respect to acidic condition), indicating an interaction of N(7) atom with Au less strong than in Au−guanine NPs. On the contrary, the H1′−H5′ sugar proton signals are broadened, and H-1′ and H4′ signals are significantly upfield shifted (δ H-1′ = 0.27 ppm; δ H-4′ = 0.16 ppm) compared with the values of 3 at the same pH (Figures S4.4.1−S4.4.2). The variations of ribose suggest eventual changes in its conformation upon interaction with Au. This fact stimulated us to a more detailed investigation by using ATR-FTIR spectroscopy (see below). 1 H NMR spectrum of Au−guanosine-5,monophosphate(4) NPs (Figures S4.5.1−S4.5.3) shows a downfield shift of the H-8 signal (δ = 0.49 ppm, with respect to the acidic condition). The H1′−H4′ signals of ribose are broadened and slightly downfield, and only the H5′ signal undergoes a really significant downfield change (δ = 0.15 ppm), likely due to its proximity to the phosphate group. Indeed, 31P NMR revealed a δ P value drastically upfield (from 2.87 to 0.34 ppm) upon formation of Au NPs, indicating a strong interaction with Au. The interactions of 5′-GMP and gold occurs throughout guanosine N(7) atom and oxygen atom(s) of phosphate group, as already reported elsewhere in metal coordination complexes.36 1 H NMR spectra of Au−1-methylguanosine (5) NPs reveal only little changes and broadening of ribose signals (Figures S4.6.1−S4.6.2). The presence of the methyl substituent in N(1) does not provide information on the coordination of 5 to Au NPs, but NMR data suggest that ribose conformation is not affected by the presence of gold. On the contrary, interesting information can be obtained by 1 H NMR spectrum of Au−8-bromo-guanosine (6) NPs, in which strongly upfield shifted signals of H3′−H5′protons have been observed (δ = 0.1−0.28 ppm). The H1′ and H2′ signals even disappear from the usual region (Figures S4.7.1−S4.7.2). Taking into account that substituted nucleosides (and nucleotides) exist mainly as syn conformations, the torsional angle of the glycosidic bond in 5 can induce different ribose conformations to fulfill the interaction with Au surface. Of course no information is obtained for H8 signal, being H substituted by a Br atom. In 1H NMR spectrum of Au−inosine (1) NPs, a large number of signals appeared in the 8.4−8.2 ppm region making it more complicated, probably because of 1 oligomer species formation, and/or side products. (Figures S4.2.1−S4.2.2). In the case of inosine, the electron density on aromatic rings does not allow a simple and univocal coordination of 1 (or oligomers) to Au surface, with N(1) and N(7) both being prone to binding with gold.

expanded table is reported in the Supporting Information, Table S4.1). Table 2. 1H and 31P Chemical Shifts of Free (1−6) Lligands at pH 3.5 and after Interaction with AuNPs mult H2 H8 H1′ H2′ H3′ H4′ H5′ H5′ H8 H8 H1′ H2′ H3′ H4′ H5′ H5′ H8 H1′ H2′ H3′ H4′ H5′ P H8 H1′ H2′ H3′ H4′ H5′ H5′ CH3 H1′ H2′ H3′ H4′ H5′ H5′

Δ

Ino (1) s 8.24 s 8.12 d 6.00 buried under H2O dd 4.36 dd 4.20 dd 3.85 dd 3.77 Gua (2) s 7.98 Guo (3) s 7.96 d 5.86 buried under H2O dd 4.35 q 4.17 dd 3.82 dd 3.76 5′-GMP (4) s 8.09 d 5.85 buried under H2O dd 4.42 q 4.25 dd 3.96 s 2.87 M1G (5) s 7.92 d 5.85 buried under H2O dd 4.35 q 4.17 d 3.82 d 3.76 s 3.41 8-Br-Gua (6) s 5.81 dd 4.91 dd 4.28 q 4.17 d 3.82 d 3.75

mult Au−Ino s s d

Δ 8.42−8.19 8.42−8.19 6.20−6.07

signal dd dd dd dd Au−Gua s Au−Guo s d

4.41 4.25 3.96 3.81 8.28 7.90 5.59

signal dd q dd dd Au−5′-GMP s d

4.40| 4.01 3.82 3.65

dd q dd s Au−M1G s d

4.42 4.32 4.11 0.34

8.58 5.86

signal

7.99 5.88

signal dd 4.39 q 4.24 d 3.84 d 3.82 s 3.46 Au−8-Br-Gua s dd dd 4.15 q 3.95 d 3.73 d 3.68

At pH 3.5, the H-8 signal of guanine (2) moves downfield as the N(7) atom protonation causes electron density to be withdrawn from H-8, in accordance with α data. The proton resonances of inosine (1) as well as of the (3−6) purine derivatives are not much affected by acidic pH and do not change considerably. On the contrary, the phosphate moiety suffers by the acidic medium, and the value of 31P chemical shift of 5′-GMP did change from 3.96 to 2.87 ppm with pH moving from physiological to 3.5. The change can be attributed to the monoprotonation of an oxygen anion (pKa ∼ 6.3).40 Analyzing 1H NMR spectrum of Au−guanine (2) NPs, we observed the H-8 signal at 8.27 ppm, shifted downfield respect 3006

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The conclusion derived from NMR results highlighted that guanine N(7) atom is involved in the interaction with gold surface, while the ribose moiety seems free from the binding, undergoing some conformational rearrangements (Scheme 1).

Scheme 2. Schematic Representation of the Conformational Changes of the Ribose Moiety after Binding to the AuNPs: (A) Guanosine, (B) Guanosine-5′-monophosphate, (C) Inosine, and (D) 8-Bromoguanosine

Scheme 1. Schematic Pictorial Representation of the Binding Sites between Guanine, Guanosine, and Guanosine 5′Monophosphate and Gold Nanoparticle Surface

ATR-FTIR Data. We performed ATR-FTIR analyses to understand if other groups could be involved in the Au−ligand interaction and to have a clearer view of the changes of the ribose puckering. In this context, bands of amino- and carbonyl stretching vibrations are of interest, as well as ring breathing vibrations of guanine moiety, that are useful to diagnose the ribose conformation; in fact, the ring vibrations result shifted by ribose−guanine vibration couplings, which, in turn, depend on both the ribose conformation and the guanine−ribose linkage.22 In fact, nucleosides (and nucleotides) can have different conformations in dependence on the various torsional angles, from an extended structure (with the phosphate group stretched away from the base) to a compact one (with phosphate, ribose, and base lumped together). For instance, in guanosine 5′-monophosphate the rotation around the N(9)− C(1′) glycosidic bond brings the C(2) carbon of the base away from the ribose (anti; α = −30°) or toward the ribose (syn; α = +150°).24 So the ribose moiety can mainly assume two different conformations: N (C3′-endo-C2′-exo) and S (C2′-endo-C3′exo) (see later Scheme 2). However, it was found elsewhere that anti-S conformation is predominant for guanosine 5′monophosphate.21 The S5 paragraph in Supporting Information includes all the details of FTIR spectroscopy applied to our systems. It reports (1) the spectra of free 1−6 compounds (black line) and Au− 1−6 NPs (red line); (2) the tables of the most important data for each spectrum; and (3) the attributed vibration values. Moreover, with a few individual bands being strongly overlapped, for better resolution we used the second derivative spectra, having a maximum at the same position as the original band, but smaller bandwidth.41 In the IR spectrum of Au−guanine (2) NPs (Figure S5.2.1), a strong band at 3202 cm−1, attributed to the stretching modes of the N(2)-H2 group, is red-shifted of 119 cm−1, suggesting its vertical orientation toward Au. The C(6)O stretching decreased in intensity and shifted at lower frequencies with respect to 2, indicating an elongation of C(6)O bond, and, therefore, its interaction via the oxygen lone pair or the double bond. The drastic weakness of CO stretching vibration

suggests a disposition of the double bond nearly parallel to Au surface, while the enhanced out of plane C−H bending indicates the tilting of 2 toward gold. In addition, analyzing the second derivative spectrum of Au−guanine (2), a broad and intense peak, comprising ν(N(7)−C(8)) and ν(C(5)−N(7)), appears after interaction (Figure S5.2.2), pointing out the effect on the coordination on the five member ring.34 Correlating NMR and FTIR data, we can conclude that guanine interacts with gold via its N(7) lone pair and CO double bond, being arranged in a bent position toward Au surface. The spectrum of Au−guanosine (3) appears to be significantly different from that of free guanosine, because of the nanoparticle-induced changes in the structure of 3 (Figure S5.3.1). Notably, the N(2)−H2 stretching vibrations almost disappear, while a very weak band at 2923 cm−1 is detectable, tentatively attributed to a hydrogen bond between Au and a H−N group of 3. A strongly shifted and split band arises from the carbonyl group, confirming its participation in the interaction with Auδ+ gold sites. On the other hand, a sharp and intense signal, attributed to N−H bending, changes by 71− 95 cm−1 compared to that of 3, due to the redistribution of electronic density in the aromatic part, involving the perturbations of N(1)−H and N(2)−H2 bonds, which can originate H-bindings with negatively charged gold centers. Interestingly, both the ribose C−C and guanine ring breathing vibration bands undergo a significant change, being shifted at 3007

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Figure 4. ATR-FTIR spectra of free 5′-GMP (black line) and Au-5′-GMP NPs (red line) in the 3500−600 cm−1 range. 31P NMR spectra of free 5′GMP at pH 3.5 (a) and Au-5′-GMP NPs (b) in the 4.5−0.0 ppm range.

lower wavenumbers upon interaction with Au, suggesting the changes in the ribose conformation: a C3′-endoanti versus O4′ -endoanti,20 according to the literature data34 (Scheme 2, panel A). Correlating NMR and IR results we can conclude that 3 interacts with the gold surface throughout N(7) and CO moieties, reinforced by hydrogen bonds carried out by NH groups and hydroxyl substituents of a conformational different ribose. The FTIR spectrum of Au−guanosine-5′-monophosphate (4) (Figure 4, red line) shows the guanine moiety of 5′-GMP involved in the interaction with gold in a similar way: a middleintensity C(6)O stretching band splits into two shoulders and shifts of 32 cm−1. Moreover, the shift of 16 cm−1 of the phosphate band is related to its different spatial disposition after interaction with Au, being in a monoanionic form, i.e., HO− P(O)(−O−R)(−O− (R = nucleoside). A sharp band at 3334 cm−1, assigned to ν (OH) of the ribose, suggests its proximity to the gold surface, while its puckering changes assuming a C1′ exoanti conformation21 (Scheme 2, panel B), allowing an easy approach of phosphate group. The 31P NMR spectrum (Figure 4) is in agreement with FTIR results. FTIR spectrum of Au−1-methylguanosine (5) NPs (Figure S5.5.1) shows new vibrations with respect to free 5. We observed a sharp band at 3214 cm−1, arising from NH2 stretching, and a redistribution of intensities of the components in the 1700−1540 cm−1 region: CO stretching band becomes less intense and shifts of 42 cm−1, while the NH2 bending band takes part of a broad and intense peak, that includes also a two-shoulder band of N(7)−C(8) and C(5)− N(7) vibrations. These pieces of evidence indicate the M1G compound to interact with Au via CO double bond, besides the N(7) lone pair (NMR evidence), and suggest that the plane of 5 is slightly bent toward the gold surface, confirming the general tendency of disposition of 1−6 compounds on the Au NPs. The ribose (C−O) stretching modes decrease in intensity, becoming hardly detectable, at the same time being well distinguishable in the second derivative spectrum. Interestingly, the band of guanine ring breathing remains centered at 670 cm−1, indicating the conservation of ribose C3′- endoanti conformation in good agreement with NMR data.21

FTIR spectrum of Au−inosine (1) NPs shows large differences of the signals in comparison with the free 1 (Figure S5.1.1): a new weak band at 3425 cm−1 was assigned to OH stretching of ribose moiety, whereas N(1)−H stretching band is shifted of 80 cm−1 at lower frequencies. The C(6)O band is split into two components of low intensity, while N(1)−H bending and N(7)−C(8) ring stretching bands are stuck together forming an intense band, respectively. It probably means that inosine has nearly an edge-on disposition on the gold surface, being also able to get involved in CO interaction and a hydrogen bond with N(1)−H. Considering ribose vibrations, we observed a sharp band at 1204 cm−1, slightly shifted in comparison with 1, and C−C ribose ring vibrations decreased in intensity. The position of 1 ring breathing bands does mean a C3′,C4′ exosyn conformation of ribose moiety becoming a C3′-endo-anti when bound to the gold.21 The correlation of NMR and FTIR data of Au−inosine NPs suggests that Au−1 NPs behave differently in D2O (1H NMR) and in the solid state (ATR-FTIR). In solution, the ligand shell is probably not stable with oligomer species being detached and free in D2O, while in the solid phase the interactions between inosine and gold can be detected, suggesting its edge-on disposition on the gold surface, involving an electronic donation of C(6)O oxygen lone pair coupled with an hydrogen bond of N(1)−H. Finally, the stretching vibrations of N(1)−H and NH2 of free 8-bromoguanosine (6) become a large shapeless band in Au−6 NPs (Figure S5.6.1), composed of several signals, whereas the CH stretching decreases in intensity and shifts. Moreover, a strong peak appears in the 1700−1500 cm−1 region, including both CO stretching and bending vibrations of N(1)−H and NH2. In particular, a two-shoulder band at 1696−1636 cm−1 attributed to ν(CO) and NH bending band at 1518 cm−1 are both shifted to lower wavenumbers (see the second derivative spectrum for a better examination). The ribose vibrations give a new large band composed of two shoulders at 1079 and 1032 cm−1, and C−C ribose vibrations at 819 and 731 cm−1. Notably, the C2′-endosyn ribose conformation in free 6, changes to C3′-endosyn.21 The correlation of NMR and FTIR data indicates the 8-bromoguanosine coordination to the gold particles via N(7) lone pair is not favored by the steric 3008

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magnitude evaluation of the upper limit of the number of adsorbed molecules, we assume that (a) the AuNPs are monodisperse; (b) guanine compounds may stick to the surface with any of its three inertial vectors perpendicular to the surface; (c) guanine compounds occupy, around the AuNPs, a spherical shell of volume Vshell whose thickness is equal to the molecular dimension perpendicular to the surface, and whose space occupation factor is 0.7 (Supporting Information, Figure S3.2). On the basis of the previous assumptions and the calculations (see Supporting Information), Table 4 reports the number of

hindrance of Br substituent. The Au−6 NPs are therefore stabilized by the electronic interactions of CO double bond and NH2. Due to the Br atom, the ribose puckering assumes a C3′-endosyn conformation, the same observed in the free ligand (Scheme 2, panel D). On the basis of the above interpretation of NMR and FTIR results, we can conclude that 1−5 compounds interact with the particles via N(7) atom and CO group, in the neutral form, as the H-8 signal is shifted in 1H NMR and the CO band changed in ATR-FTIR spectra, in comparison with free compounds. Br-guanosine (6) differs in its binding, while inosine (1) gives more than one product in the colloidal system. Moreover, guanosine and 8-Br-guanosine derivatives wre shown to be able to form weak hydrogen bonds between N(1)−H group and gold surface, having observed changes of stretching and bending NH bands after interaction with gold. This behavior was already reported by us in the case of Au−thymine (or thymidine and thymidine 5′-monophosphate).42 The substitution on C(8) atom leads to different constraints depending on bulky substituent, and different pathways of reaction can be possible for the building up of Au−purine NPs, as demonstrated for Au−Br-guanosine NPs. Novel results on the conformational change of ribose puckering highlight that it can occur when purine derivatives interact with Au nanoparticle (Table 3). The need of conformational changes is strictly

Table 4. Number of Molecules of Guanine Compounds (L) per Gold Nanoparticle Found from Theoretical Calculations (calcd) and Quantitative Analyses (exptl)a L Gua Guo 5-GMP M1-Guo 8BrGuo Ino

L per NP (calcd)

L per NP1

L per NP2

L per PN3

L per NP (exptl)

× × × × × ×

507 625 2000 339 678 154

1715 940 126 564 558 381

3850 2100 281 1285 1268 883

5302 2890 405 1776 5055 1235

168 236 71 152 150 150

9.5 9.6 1.8 1.8 1.8 3.9

1012 1012 1014 1013 1013 1013

a N (AuNP) is the number of gold nanoparticles, while 1 is the calculated number of ligands per nanoparticle considering packing coefficient 0.35 (flat orientation of the ligands), 2 is the calculated number of ligands per nanoparticle considering packing coefficient 0.75 (edge-on orientation of the ligands), and 3 is the calculated number of ligands per nanoparticle considering packing coefficient 1 (vertical orientation of the ligands).

Table 3. Ribose Puckering and Ring Breathing Bands before and after Interaction with Gold Nanoparticles guanosine (3) C3′-endoanti 686 cm−1 guanosine-5′-GMP (4) C2′-endoanti 699 cm−1 1-methylguanosine (5) C3′-endoanti 668 cm−1 inosine (1) C3′-exosyn 692; 629 cm−1 8-Br-Guo (6) C2′-endosyn 681 cm−1

N (AuNP)

Au−guanosine O4′-endoanti 665 cm−1 Au−5′-GMP C1′-exoanti 674 cm−1 Au−M1G C3′-endoanti 670 cm−1 Au−ino C3′-endoanti 662 cm−1 Au−8-Br-Guo C3′-endosyn 667 cm−1

gold nanoparticles stabilized by 1−6 guanine compounds in each studied colloidal system and stoichiometric ratios between the ligands and nanoparticles in the experimental conditions. Moreover, the number of ligands per particle was calculated, considering flat, edge-on, or vertical orientations of the ligands. The ratios in the three cases were obtained and compared to the stoichiometric numbers of ligands per particles. While the result does not really help in determining which of the three adsorption modes is favored, it suggests that the presence of multiple layers on the surface is unlikely, because the available Au surface hardly allows for full coverage, while Gu···Gu interaction energies seem smaller than Gu···Au desorption energies. If the edge-on adsorption mode is assumed, as discussed above, with the current stoichiometry only a part of the surface space is occupied and lateral Gu···Gu interactions should be sparse. This corroborates our assumption of nearly isolated Gu surface molecules. Moreover, Table 4 reports the experimental values of each ligand per particle found by quantitative analysis. By comparison of calculated and experimental values of ligands per particle we can conclude the presence of only one layer of guanine compounds covering gold surface.

related to the electronic properties of guanine ring after contact with Au particle surface throughout N(7) and CO. Moreover, the phosphate group of 4 is strongly involved in a polar interaction with gold surface, being monoprotonated, HO(O)PO−R)(O−) due to the pH of colloids. Coverage of Nanoparticles. Size parameters for guanine (and guanine compounds) can be obtained from crystallographic data (Cambridge Structural Database) along with the three inertial molecular axes. The molecular volumes of guanine compounds are constant over neutral, protonated, or tautomerized rings (Table S7.2. in Supporting Information). Guanine itself forms in its crystal slightly corrugated flat monolayers (Supporting Information, Figure S7.1) held together by later hydrogen bonds, with layer periodicities of 0.97 and 1.65 nm, stacked at the usual aromatic−aromatic distance of 0.35 nm.43 Intralayer HB pairing energies are in the range 60−70 kJ mol−1, while stacking cohesive energies are 25− 30 kJ mol−1.44,45 The overall packing factor, or the ratio of molecular volume within the cell to cell volume, is in the range 0.7−0.8 for the above-mentioned crystals. Some comments on actual coverage, based on molecular and atomic sizes and current stoichiometry, can be remarked. For an order-of-

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CONCLUSIONS We have synthesized small water-soluble gold nanoparticles modified by 1−6 purine compounds following a simple, allpurpose reduction method. The obtained sols have been shown to possess a good colloidal stability, having zeta-potential values in the range between −20 and −50 mV. The most part of compounds (1, 3−6) exists in solution in the neutral (unprotonated) form, under given experimental conditions, whereas guanine appears in both protonated and unprotonated 3009

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(13) Woffendin, C.; Yang, Z.; Udaykumar, Xu, L.; Yang, N.; Sheehy, M. J.; Nabel, G. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11581−11585. (14) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128−4158. (15) Bielinska, A.; Eichman, J. D.; Lee, I.; Baker, J. R., Jr.; Balogh, L. J. Nanopart. Res. 2002, 4, 395−403. (16) Nakao, H.; Shiigi, H.; Yamamoto, Y.; Tokonami, S.; Nagaoka, T.; Sugiyama, S.; Ohtani, T. Nano Lett. 2003, 3, 1391−1394. (17) Ostblom, M.; Liedberg, B.; Demers, L. M.; Mirkin, C. A. J. Phys. Chem. B 2005, 109, 15150−15160. (18) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125 (30), 9014−9015. (19) Sundaralingam, M. Ann. N.Y. Acad. Sci. 2006, 225 (1), 8205− 8212. (20) Remin, M.; Shugar, M. Biochem. Biophys. Res. Commun. 1972, 48 (3), 636−642. (21) Son, T. D.; Guschibauer, W. Nucleic Acids Res. 1975, 2 (6), 873−888. (22) Nishimura, Y.; Tsuboi, M.; Sato, T. Nucleic Acids Res. 1984, 12 (17), 6901−6908. (23) Bogdan, D.; Morari, C. J. Phys. Chem. C 2012, 116, 7351−7359. (24) Son, T. D.; Guschibauer, W.; Gueron, M. J. Am. Chem. Soc. 1972, 94 (22), 7903−7911. (25) Shukla, M. K.; Leszczynski. J. Chem. Phys. Lett. 2006, 429, 261− 265. (26) Ubiali, D.; Serra, C. D.; Serra, I.; Morelli, C. F.; Terreni, M.; Albertini, A. M.; Manitto, P.; Speranza, G. Adv. Synth. Catal. 2012, 354 (1), 96−104. (27) Chen, C. F.; Tzeng, S. D.; Chen, H. Y.; Lin, K. J.; Gwo, S. J. Am. Chem. Soc. 2008, 130, 824−826. (28) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. Am. Chem. Soc. 2003, 125 (17), 5219−5226. (29) Zhao, W.; Chiuman, W.; Lam, J. C. F.; Brook, M. A.; Li, Y. Chem. Commun. 2007, 36, 3729−3731. (30) Zhao, W.; Gonzaga, F.; Li, Y.; Brook, M. A. Adv. Mater. 2007, 19, 1766−1771. (31) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076−10084. (32) Leszczynski, J. Advances in Molecular Structure Research; Hargittai, M., Hargittai, I., Eds.; JAI Press: Stamford, CT, 2000; Vol. 6, p 209. (33) Skoog, D. A.; Weat, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry; Saunders: New York, 2005. (34) Pergolese, B.; Bonifacio, A.; Bigotto, A. Phys . Chem. Chem. Phys. 2005, 7, 3610−3613. (35) Kryachko, E. S.; Remacle, F. Nano Lett. 2005, 5 (4), 735−739. (36) Lippert, B. Coord. Chem. Rev. 2000, 200−202, 487−516. (37) Schimanski, A.; Freisinger, E.; Erxleben, A.; Lippert, B. Inorg. Chim. Acta 1998, 283, 223−232. (38) Colacio, E.; Crespo, O.; Cuesta, R.; Kivekas, R.; Laguna, A. J. Inorg. Biochem. 2004, 98, 595−603. (39) Yang, D.; Van Boom, S. G. E.; Reedijk, J.; Van Boom, J. H.; Wang, A. H. Biochemistry 1995, 34, 12912−12920. (40) Martin, R. B. Acc. Chem. Res. 1985, 18, 32−38. (41) Shaw, C. P.; Middleton, D. A.; Volk, M.; Lévy, R. ACS Nano 2012, 6, 1416−1426. (42) Avvakumova, S.; Scari, G.; Porta, F. RSC Adv. 2012, 2, 3658− 3661. (43) Guille, K.; Clegg, W. Acta Crystallogr., Sect. C 2006, 62, 515− 517. (44) Gavezzotti, A. Molecular Aggregation; Oxford University Press: Oxford, U.K., 2007. (45) Gavezzotti, A. New J. Chem. 2011, 35, 1360−1368.

form. The interaction of 1−6 purine derivatives with gold particles was deeply studied by 1H NMR and ATR-FTIR spectroscopy, determining the binding sites between organic compounds and gold surface: N(7) and CO groups of the guanine moiety are the major interaction sites for each molecule, while ribose-containing molecules (3 and 6) are also able to form a hydrogen bond via N(1)−H site of guanine ring. The ribose moiety does change its puckering when coordinated to gold, causing interaction of the phosphate group, in the case of guanosine 5′-monophosphate molecule.

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ASSOCIATED CONTENT

S Supporting Information *

Calculation of the concentration fraction α, table containing molar ratio between the reagents, pKa values and estimated protonation states, TEM micrographs and size distribution diagrams, UV−vis spectra, 1H and 31P NMR data, and FTIR spectra of free ligands and AuNPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.A.), francesca. [email protected] (F.P.). Phone: +390250314361. Fax. +390250314405. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Prof. Angelo Gavezzotti for his precious help on determining the nanoparticle coverage, Pasquale Illiano for NMR assistance, and Dr. Stefano Pieraccini for theoretical structural analysis. Financial support from Fondazione Cariplo (Ricerca Scientifica e Tecnologica sui Materiali Avanzati−2009-2696) is greatly acknowledged.

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REFERENCES

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