Light-Triggered Thiol-Exchange on Gold Nanoparticles at Low

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Light-Triggered Thiol-Exchange on Gold Nanoparticles at Low Micromolar Concentrations in Water Christian Franceschini, Paolo Scrimin,* and Leonard J. Prins* Department of Chemical Sciences, University of Padova, Padova 35131, Italy S Supporting Information *

ABSTRACT: The place-exchange reaction of thiol-containing peptides in a cationic monolayer on gold nanoparticles occurs very rapidly at low micromolar concentrations in water with excellent control over the degree of substitution. The driving force for this process is the binding of anionic peptides to a cationic monolayer surface which causes a strong increase in the local concentration of thiols. The place-exchange reaction can be triggered by light using a photolabile protecting group on the thiol moiety.

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INTRODUCTION Thiol monolayer protected gold nanoparticles (Au NPs) have emerged as highly attractive multivalent systems for applications in nanomedicine and diagnostics.1,2 Their attractiveness originates from a high biocompatibility and their ease of preparation and functionalization.3,4 The presence of different thiols in the monolayer results in heterofunctionalized multivalent systems. Generally, such mixed monolayers are obtained by a place-exchange reaction in which an externally added thiol displaces a thiolate from the monolayer.5,6 The place-exchange reaction has been studied in great detail, and these studies have demonstrated that thiols exchange in a 1:1 fashion through an associative “SN2-type” mechanism and that the leaving thiolate is expelled as a free thiol rather than as a disulfide.7−9 Nonetheless, the place-exchange reaction is kinetically rather slow and the extent of it depends strongly on the amount of added thiol. Consequently, place-exchange reactions are typically performed using an excess of thiol at millimolar concentrations with reaction times in the order of hours or days. In sharp contrast, we show here the instantaneous exchange of thiols in a cationic monolayer on Au NPs at low micromolar concentrations in water using a stoichiometric amount of thiol. It is also shown that the placeexchange reaction can be activated in situ by light using a photolabile thiol-protecting group. Gold nanoparticles protected with cationic monolayers (e.g., having quaternary ammonium or metal-complexes as peripheral groups) are very frequently being used in bioapplications because of their high solubility in water and their high affinity for oligo- or polyanions.10,11 We have exploited the high affinity of oligoanions, such as Asp-rich peptides and nucleotides, for cationic monolayers to develop catalytic and sensing systems.12−14 These oligoanions bind to the monolayer under saturation conditions at low micromolar concentrations in © XXXX American Chemical Society

water. This binding process is driven by a combination of (multiple) electrostatic interactions and hydrophobic interactions.15 Despite the high thermodynamic affinity, the exchange of oligoanions on the surface is kinetically very fast and exchange between two different oligoanions occurs instantaneous.16 This property is extremely useful for setting up sensing and catalytic systems, but is not compatible with in vivo applications of the self-assembled complexes. For that reason, we started to insert Cys-residues in the anionic peptides in order to explore the possibility of exploiting the far more stable Au−S interaction to increase the kinetic stability of our system. This approach was also motivated by previous reports by Rotello et al. that had shown that the place-exchange reaction between thiols and glutathione is an effective way to release DNA and proteins from the surface of cationic nanoparticles.17−19 Current knowledge lets us anticipate that the very fast, electrostatically-driven interaction of the anionic peptides with the cationic surface would be followed by a much slower thiol exchange at the gold cluster surface.

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RESULTS AND DISCUSSION i. Interaction of Thiol-Containing Peptides with Au NP 1. The interaction between Au NP 1,16,20 covered with thiol 2, and a set of small peptides (both with and without Cysresidues) was initially studied in two different ways (Figures 1 and 2d). For peptides containing a fluorescent Trp-moiety, direct fluorescence titrations to Au NP 1 were possible taking advantage of the fact that Au NPs are very effective fluorescence quenchers. In addition, displacement studies using the competing reference anionic probe A were performed for all Received: September 1, 2014 Revised: October 28, 2014

A

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Figure 1. Schematic representation of the different behavior of anionic peptides with (right) and without (left) free thiols upon addition to Au NP 1. Structure of thiol 2 and the fluorescent probes A and B.

Figure 2. (a) Fluorescence titrations of Trp-containing peptides to Au NP 1. (b) Displacement of probe A from the surface of Au NP 1 upon the addition of different peptides. (c) Fluorescence intensity of probe A as a function of time upon the addition of thiol-containing peptides. (d) Structure of the used peptides; the color codes refer to those used in panels (a)−(c). Full experimental details are available in the Supporting Information.

peptides in order to determine their relative affinity for Au NP 1 compared to A. The strong difference between a peptide with a free thiol and one without emerged immediately from a comparison of the fluorescence titrations of Ac−CWD−OH and Ac−WD−OH to Au NP 1 (≈ 150 μM) (Figure 2a). Previously, we had shown that anionic peptides bind to the surface of a cationic

nanoparticle with a strength directly related to the number of negative charges on the peptides.12 In line with these results, a rather low affinity of Ac−WD−OH for Au NP 1 was observed as evidenced by the shallow binding isotherm. In sharp contrast, however, Ac−CWD−OH bound under saturation conditions exhibiting a surface saturation concentration of 22.5 μM. Considering the same overall charge of the peptides, this B

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Figure 3. Ultrafiltration experiments followed by LC/MS. For (Ac−CW−OH)2 (a) and Ac−CW−OH (b), the SIM-channels corresponding to the respective peptides (red) and thiol 2 (blue) of the chromatograms are given for 3 phases: I, free peptide; II, after the addition of Au NP 1; III, after the addition of probe A. The gray peak marked with an asterisk is an impurity originating from the PES-membranes (see the Supporting Information). Full experimental details are given in the Supporting Information.

gave a nearly instantaneous increase in the fluorescence intensity which then remained constant indicating that the exchange process with A occurs very rapidly. These studies indicate two features. First, the presence of a Cys-residue in these peptides is a prerequisite for the displacement of A. Second, the different displacement levels observed for Ac− CWD−OH, Ac−CW−OH, Ac−C−OH, and Ac−C−NH2 indicate the importance of the number of charges present in the thiol-peptide. ii. Monitoring of the Place-Exchange Reaction through Ultrafiltration. The best explanation for the above observations is that binding of the thiol-peptides to the monolayer surface is followed by a rapid place-exchange reaction with thiol 2. However, the fluorescence experiments do not provide information on whether this is indeed the case. Typically, the place-exchange reaction is monitored by 1H NMR spectroscopy, but the low concentrations used in this study do not permit the use of that technique. Also, thiol 2 does not contain a functional moiety that would permit detection of its release by other spectroscopies, such as fluorescence8,9 or EPR.22 A molecular insight in the exchange process was obtained from ultrafiltration experiments followed by LC-MS. As we have shown elsewhere, the use of PES-membranes with a 10 kDa cutoff permits a separation of free and nanoparticle bound molecules.23 Analysis of the dialysate by means of LCMS thus permits a straightforward analysis of any unbound molecules. In these experiments, we focused on peptide Ac−

difference came as a surprise. The observation that peptide Ac− C(tBu)WD−OH, in which the thiol-moiety is protected as tertbutyl ether, behaved similar as Ac−WD−OH indicated that the free thiol-group is responsible for this difference (Figure 2a). The difference between Ac−CWD−OH and Ac−WD−OH emerged in an even stronger way from competition experiments with probe A. Previous studies had shown that probe A has an extraordinary high affinity for cationic monolayers resulting from the presence of three phosphate groups and the hydrophobic N-methylanthraniloyl (MANT) substituent, which penetrates into the hydrophobic part of the monolayer.15 Indeed, the addition of up to 80 μM Ac−WD−OH or Ac− C(tBu)WD−OH to a solution containing Au NP 1 (≈150 μM) and probe A (10 μM) did not result in any displacement of A (Figure 2b). In sharp contrast, the addition of Ac−CWD−OH resulted in a strong increase in the fluorescence intensity originating from A (Figure 2b). The observed intensity of 580 au after the addition of 80 μM Ac−CWD−OH corresponded to nearly quantitative release of A (95%). Strikingly, even the addition of Ac−CW−OH and Ac−C−OH, having, respectively, just 2 and 1 negative charge, resulted in significant displacements of A (Figure 2b). On the other hand, neutral compound Ac−C−NH2 caused only a minor displacement of A even at 80 μM.21 Kinetic measurements were performed to study the rate of exchange by measuring the change in fluorescence intensity as a function of time upon the addition of free thiol-peptides (50 μM) to Au NP 1 covered with A (Figure 2c). All peptides C

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Figure 4. (a) Schematic representation of light-triggered thiol exchange on Au NP 1. (b) Irradiation of Ac−C(Coum)−OH 3 results in the formation of Ac−C−OH. (c) Displacement of probe B on Au NP 1 by Ac−C(Coum)−OH (circles) and Ac−C−OH (filled squares). The black square represents the fluorescence intensity measured after irradiation of a solution containing Au NP 1, probe B, and Ac−C(Coum)−OH. (d) Chromatograms of the dialysate (SIM detection of thiol 2) of a solution containing Au NP 1 and Ac−C(Coum)−OH before and after irradiation for 15 min. Full experimental details are available in the Supporting Information.

Ac−CW−OH displaces thiol 2 from the monolayer forming a Au−S bond. This unambiguously explains that the apparent high affinity observed for the thiol-peptides originates from a very fast and efficient place-exchange reaction of these peptides with thiol 2. The place-exchange process was quantitatively investigated by repeating the ultrafiltration experiments adding increasing amounts of Ac−CW−OH (5−50 μM) to Au NP 1 (in the presence of probe A) and measuring the signal intensities of both thiol 2 and Ac−CW−OH. A plot of the concentration of the thiols in the dialysate as a function of the concentration of Ac−CW−OH gave a curve for Ac−CW−OH that is almost superimposable with that obtained from the direct fluorescence titration (Supporting Information). Interestingly, an increase in the concentration of free thiol 2 in the dialysate was observed until the surface saturation concentration (SSC) of Ac−CW−OH for Au NP 1 was reached (24 μM) after which it leveled off at a concentration of around 22 μM. This experiment indicates that Ac−CW−OH and 2 exchange in a 1:1 ratio, which is in line with conclusions reported in the literature. It is further noted that the disulfide of 2 or the heterodisulfide formed between 2 and Ac−CW−OH were never observed. Compared to other systems, the place-exchange reaction between Cys-containing peptides and Au NP 1 is characterized by several surprising features. The reaction occurs at low micromolar concentrations in aqueous buffer, is very rapid, and exploits stoichiometric amounts of thiols. The latter characteristic gives a precise control over the amount of peptide that can

CW−OH and its oxidized form, the disulfide (Ac−CW−OH)2. In order to permit an accurate analysis of all possible molecules, ESI-MS in SIM-mode was used for detection. Quantification of the concentration of certain key molecules was performed by comparing the signal intensities with the respective calibration curves. A full list of all screened molecules (including the thiol 2 and possible disulfides) with their corresponding masses is provided in the Supporting Information. Ultrafiltration of a mixture of Au NP 1 and disulfide (Ac− CW−OH)2 at a concentration of 5 μM resulted in a complete disappearance of the (Ac−CW−OH)2 peak indicating its quantitative localization on the surface of Au NP 1 (Figure 3a, II). However, the addition of the competing probe A (5 equiv) resulted in a reappearance of the peak corresponding to 57% of the expected value indicating that (Ac−CW−OH)2 was bound to Au NP 1 in a reversible manner (Figure 3a, III). The observed value was in fine agreement with the value of 60% obtained from the fluorescence displacement assay (Supporting Information). Also ultrafiltration of a mixture of Au NP 1 and thiol Ac−CW−OH (10 μM) resulted in the complete absence of the Ac−CW−OH peak (Figure 3b, II), but, at difference, in this case the addition of probe A to this mixture did not result in its reappearance (Figure 3b, III). Importantly, an analysis of the other SIM-channels showed the appearance of a new intense peak corresponding to thiol 2 originating from the monolayer of Au NP 1 (Figure 3b, II + III). This peak was not observed at all for the analogous experiment using the disulfide (Figure 3a, II + III). This observation clearly demonstrates that D

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cence intensity (311 au) is in excellent correspondence with the expected value of 305 au for Ac−C−OH at 18 μM (considering the 60% yield of the photocleavage reaction). Ultrafiltration experiments indeed confirmed the release of thiol 2 after irradiation, proving that the place-exchange reaction had occurred (Figure 4d).

be inserted in the monolayer. It is important to notice that binding of the peptides to the cationic surface is a key prerequisite for thiol-exchange to occur in the micromolar concentration regime and with the observed high rate. Indeed, binding of the thiol-peptides at the monolayer surface leads to an enormous increase in effective concentration of thiol at the surface which is probably the main reason for the fast placeexchange reaction.24 The thiol-exchange may be further facilitated because of the higher local pH at the monolayer which favors deprotonation of the thiol.13 At first sight, the results presented here seem to contrast previous data obtained by Rotello and coworkers on related systems.17−19 In these studies, the place-exchange reaction between thiols of a cationic monolayer and glutathione was used as a tool to release DNA and proteins that were electrostatically bound to the nanoparticle. It was observed that this place-exchange reaction occurred efficiently at millimolar concentrations of glutathione, but not at micromolar concentrations. It is noted, though, that in these studies highly competitive species (DNA and proteins) needed to be displaced. From the displacement studies described above (Figure 2b,c), it emerges clearly that the efficacy of the place-exchange reaction strongly depended upon the relative binding affinity between thiol-peptide and probe A. Based on this observation, it is indeed expected that the displacement of multivalent competitors as in Rotello’s work required millimolar concentrations of glutathione. iii. Control over the Place-Exchange Reaction by Light. For our purposes, the thiol-exchange provides for an unique opportunity to increase the kinetic stability of the system, as this leads to a direct connection of the thiol-peptide to the gold core through a very stable Au−S bond.25 However, in order not to lose the advantages of a kinetically labile system, it is important that the insertion of the peptides in the monolayer can be triggered by an external stimulus (Figure 4a). The application of the stimulus would result in a “covalent capture” of the electrostatically bound peptides. In order to explore this possibility, we synthesized compound Ac− C(Coum)−OH 3 in which the thiol-moiety is protected with a photolabile protecting group (Figure 4b). Previous literature reports had shown that analogous compounds are effectively cleaved upon irradiation at the absorption maximum of the coumarin moiety liberating the free thiol.26,27 Photophysical studies revealed that irradiation of 3 at 400 nm effectively liberated Ac−C−OH with a yield of around 60%, which is similar to the yields reported in the literature (Supporting Information).27 Although the yield is far from quantitative, we decided anyway that this was good enough for a proof-ofprinciple study, because the side product is a thioether with a low affinity for Au NP 1.27 Kinetic studies revealed the complete disappearance of Ac−C(Coum)−OH after irradiating for 5 min at 400 nm or for 15 min at 365 nm (Supporting Information). A fluorescence titration revealed that Ac− C(Coum)−OH did not bind to Au NP 1 (Supporting Information). The low affinity of Ac−C(Coum)−OH for Au NP 1 was further confirmed by the observation that the addition of up to 30 μM Ac−C(Coum)−OH to Au NP 1 and probe B hardly resulted in the displacement of B (Figure 4c).28,29 On the other hand, the addition of 30 μM Ac−C−OH resulted in the efficient displacement of B (Figure 4c). We then irradiated the sample containing Au NP 1, probe B, and Ac− C(Coum)−OH at 365 nm for 15 min and observed a strong increase in the fluorescence intensity of probe B (Figure 4c and Supporting Information Figure S26). The measured fluores-

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CONCLUSIONS In conclusion, we have shown that the place-exchange reaction of thiol-containing peptides in a cationic monolayer on Au NPs occurs very rapidly at low micromolar concentrations in water with excellent quantitative control. The place-exchange reaction can be activated by light using a photocleavable protecting group on the thiol. This approach may serve as a “covalent capture” tool for the controlled synthesis of heterofunctionalized nanoparticles.

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ASSOCIATED CONTENT

S Supporting Information *

Materials and methods, experimental procedures, and details. Control experiments. This material is available free of charge via the Internet at This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the ERC (StG-239898) and FIRB (RINAME-RBAP114AMK) is acknowledged. REFERENCES

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