Paolo Scrimin

Exploiting the Self-Assembly Strategy for the Design of Selective CuII Ion Chemosensors

Simply by mixing in water, a liphophilic dipeptide (L), a surfactant (S), and a fluorophore (F) self-assemble to give a sensor able to detect Cu(II) ions (see scheme). Despite the ease of construction, the sensor displays high selectivity and a low detection limit for the target ion. This new modular approach to sensing devices allows easy variations of the components, making optimization of the system very simple and fast.

Phosphate Diester and DNA Hydrolysis by a Multivalent, Nanoparticle-Based Catalyst

2-nm gold nanoclusters coated with Zn(II) complexes bearing auxiliary hydrogen bond donors act as multivalent catalysts capable of promoting the hydrolysis of model phosphate diesters with exceptional activity and inducing DNA double strand cleavage.

Functional gold nanoparticles for recognition and catalysis

Gold nanoparticles passivated by a monolayer of functional thiolates represent a new promising tool in supramolecular chemistry since they allow the preparation of complex, self-organized, polyvalent structures with a very modest synthetic effort. These systems have sizes comparable to those of many biological molecules including proteins and nucleic acids, and to those of many cellular sub-structures. In this way they are good candidates to model recognition processes and to develop new biomimetic catalysts. Some interesting examples towards these goals are reviewed briefly.

Self-assembly of a catalytic multivalent peptide-nanoparticle complex.

Catalytically active peptide-nanoparticle complexes were obtained by assembling small peptide sequences on the surface of cationic self-assembled monolayers on gold nanoparticles. When bound to the surface, the peptides accelerate the transesterification of the p-nitrophenyl ester of N-carboxybenzylphenylalanine by more than 2 orders of magnitude. The gold nanoparticle serves as a multivalent scaffold for bringing the catalyst and substrate into close proximity but also creates a local microenvironment that further enhances the catalysis. The supramolecular nature of the ensemble permits the catalytic activity of the system to be modulated in situ.

The First Water-Soluble 310-Helical Peptides

Two water-soluble 3(10)-helical peptides are synthesized and fully characterized for the first time. The sequence of these terminally blocked heptamers comprises two residues of the Calpha-trisubstituted alpha-amino acid 2-amino-3-[1-(1,4,7-triazacyclononyl)]propanoic acid and five residues of a Calpha-tetrasubstituted alpha-amino acid (either alpha-aminoisobutyric acid or isovaline). Using CD and NMR techniques we were able to show that both heptapeptides are well structured in water, and that the type of conformation adopted is indeed the ternary 3(10)-helix.

Dissipative self-assembly of vesicular nanoreactors

Dissipative self-assembly is exploited by nature to control important biological functions, such as cell division, motility and signal transduction. The ability to construct synthetic supramolecular assemblies that require the continuous consumption of energy to remain in the functional state is an essential premise for the design of synthetic systems with lifelike properties. Here, we show a new strategy for the dissipative self-assembly of functional supramolecular structures with high structural complexity. It relies on the transient stabilization of vesicles through noncovalent interactions between the surfactants and adenosine triphosphate (ATP), which acts as the chemical fuel. It is shown that the lifetime of the vesicles can be regulated by controlling the hydrolysis rate of ATP. The vesicles sustain a chemical reaction but only as long as chemical fuel is present to keep the system in the out-of-equilibrium state. The lifetime of the vesicles determines the amount of reaction product produced by the system.

Detection of Enzyme Activity through Catalytic Signal Amplification with Functionalized Gold Nanoparticles

The detection of low levels of proteins and other biomarkers is of crucial importance for the early diagnosis of diseases. The development of chemical-sensing methodologies as an alternative to biological assays is of strong current interest, because such methods involve simple detection protocols. In addition, such systems can be adapted through straightforward structural modifications for use with a wide variety of targets. Nevertheless, a common feature of these assays is that the amount of generated signal is proportional to the amount of substrate converted by the enzyme. The sensitivity of such assays would be significantly increased if the enzymatic conversion of a single substrate molecule led to the formation of a multitude of reporter molecules through a cascade of chemical events, each of which magnified the previous event. Examples of chemical systems able to amplify originally weak input signals have been reported. Herein, we report the application of a catalytic amplification process for the detection of proteases. The strategy relies on a cascade of two catalytic events for signal generation, whereby a gold nanoparticle covered with a catalytic self-assembled organic monolayer (Au-MPC) has a crucial central role. In the first event, an enzyme hydrolyzes a peptide substrate, which acts as an inhibitor for the catalytic monolayer (Figure 1). Upon hydrolysis, the catalytic activity of the monolayer is restored, and large quantities of a yellow reporter molecule are produced. The Au nanoparticles are important for two reasons. First, they enable the facile, spontaneous formation of dinuclear catalytic sites on the periphery of the monolayer. Second, their multivalent nature permits the occurrence of multipoint interactions with (biological) targets. The latter aspect, together with the intrinsic physical and chemical properties of the nanoparticles and the ease of their preparation and functionalization, has led to extensive development of assays based on Au nanoparticles that also occasionally rely on various forms of signal amplification. Previously, we showed that Au-MPC 1 catalyzes the transphosphorylation of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNPP) highly efficiently. HPNPP is an activated RNA model substrate. Detailed kinetic studies revealed that catalysis results from the cooperative action of two triazacyclononane·Zn (TACN·Zn) complexes localized on the surface of the monolayer. 22] Au-MPC 1, which is fully covered with the TACN·Zn complex, displays enzymelike saturation behavior with the “overall” values kcat = 6.7 10 3 s 1 and KM = 0.31 mm at pH 7.5 in H2O. [23] This system is intriguing for the following reasons: a) under the experimental conditions, there is practically no background reaction, since kuncat under the same conditions is of the order of 10 7 s ; b) the reaction can be monitored visibly by measuring the absorbance of the p-nitrophenol product at 400 nm; c) a surprisingly high affinity is observed for the binding of HPNPP to 1. Since Au-MPC 1 has a multitude of positively charged TACN·Zn complexes on its surface, we anticipated that the system would have a high affinity for oligoanions owing to multivalent interactions. This hypothesis was also supported by the contributions by Hamachi and co-workers, who demonstrated that oligophosphates and oligoaspartates bind a bis(zinc(II) dipicolylamine) complex with very high affinity. In our system, such oligoanions would act as competitive inhibitors for HPNPP and thus turn off the catalytic activity of the system. To verify whether we could use the catalytic production of p-nitrophenol as a tool to detect binding events on the AuMPC surface, we studied two series of biologically important oligoanions (peptides and phosphates) with negative charges increasing from 1 to 4. The peptide series comprised BocNHGly-OH (1 ), AcNH-Asp-OH (2 ), AcNH-Asp-Asp-OH (3 ), and AcNH-Asp-Asp-Asp-OH (4 ), and the phosphate series cAMP (1 ), AMP (2 ), ADP (3 ) and ATP (4 ; Figure 1). Increasing amounts of each compound were added to a solution of Au-MPC 1 in H2O buffered at pH 7.0 at 40 8C with the concentration of TACN·Zn headgroups equal to 5 mm. This value implies a Au-MPC 1 concentration of around 100 nm, on the basis of the knowledge that a 1.6 nm sized nanoparticle contains roughly 50 thiols. A kinetic Zn titration confirmed that at these concentrations, the Zn ions are quantitatively bound to the TACN ligand (see the Supporting Information). Subsequently, HPNPP was added to give an initial concentration of 1 mm in the mixture, and the initial rate of cleavage, ninit, was measured for 30 min by monitoring the increase in absorbance at 400 nm. A plot of the initial rate (n1), normalized with respect to the initial rate in the absence of an inhibitor (n0), as a function of the concentration of added inhibitor, gave the inhibition curves depicted in Figure 2 for the peptides (the data for the [*] Dr. R. Bonomi, A. Cazzolaro, Dr. A. Sansone, Prof. Dr. P. Scrimin, Dr. L. J. Prins Department of Chemical Sciences University of Padova Via Marzolo 1, 35131 Padova (Italy) Fax: (+ 39)049-827-5239 E-mail: paolo.scrimin@unipd.it leonard.prins@unipd.it Homepage: http://www.chimica.unipd.it/leonard.prins/pubblica/

Expeditious synthesis of water-soluble, monolayer-protected gold nanoparticles of controlled size and monolayer composition.

A protocol is reported for the preparation of water-soluble, thiol-protected Au nanoparticles (Au-MPC) where dioctylamine is used as a stabilizing agent when the gold cluster is formed using the two-phase Brust and Schiffrin procedure. The amount of amine controls the size of the nanoparticles in the 1.9-8.9 nm diameter range. The final stabilization of the gold clusters by addition of functionalized thiols is performed under very mild conditions compatible with most biomolecules. The procedure is suitable for a wide variety of functional groups present in the thiol and allows one to use thiol mixtures with a precise control of their composition in the monolayer. As a proof of principle, examples of nanoparticles protected with thiols comprising functional groups ranging from polyethers, saccharides, polyamines and ammonium ions are reported.

Synthesis, characterization and properties of water-soluble gold nanoparticles with tunable core size

Robust, water-soluble gold clusters protected by monolayers of ligands containing a short alkyl chain (C7) close to the gold surface and a triethylene glycol monomethylether unit (TEG) to impart solubility in water and other polar solvents were prepared and characterized. Thiol 7 (N1-{2-[2-(2-methoxyethoxy)ethoxy]ethyl}-8-sulfanyloctanamide) constitutes a good and versatile capping agent for the preparation of these nanoparticles. By tuning the Au/thiol ratio and sodium borohydride addition rate, nanoparticles with different core diameters ranging from 1.5 to 4.2 nm, as determined by TEM analysis, could be obtained. The size distribution of the gold cores appears to become broader as the Au/thiol ratio used to prepare the nanoparticles increases. Characterization of these nanoclusters also by NMR, UV-Vis and FTIR spectroscopies is reported. Solubility properties have been studied in a large variety of solvents and different solubility behaviors were observed for nanoparticles of different sizes. Exchange reactions were carried out successfully with small (1.9 nm) and large nanoparticles (4.2 nm) using dodecanethiol as the entering thiol. This demonstrates that these materials can be used for the preparation of nanoclusters with different functional groups soluble in polar solvents including water. The synthetic procedure described represents a facile route to tailoring the size and solubility properties of Au nanoparticles.

Catalytic self-assembled monolayers on Au nanoparticles: the source of catalysis of a transphosphorylation reaction.

The catalytic activity of a series of Au monolayer protected colloids (Au MPCs) containing different ratios of the catalytic unit triazacyclononane⋅Zn(II) (TACN⋅Zn(II) ) and an inert triethyleneglycol (TEG) unit was measured. The catalytic self-assembled monolayers (SAMs) are highly efficient in the transphosphorylation of 2-hydroxy propyl 4-nitrophenyl phosphate (HPNPP), an RNA model substrate, exhibiting maximum values for the Michaelis-Menten parameters k(cat) and K(M) of 6.7×10(-3) s(-1) and 3.1×10(-4) M, respectively, normalized per catalytic unit. Despite the structural simplicity of the catalytic units, this renders these nanoparticles among the most active catalysts known for this substrate. Both k(cat) and K(M) parameters were determined as a function of the mole fraction of catalytic unit (x(1)) in the SAM. Within this nanoparticle (NP) series, k(cat) increases up till x(1) ≈0.4, after which it remains constant and K(M) decreases exponentially over the range studied. A theoretical analysis demonstrated that these trends are an intrinsic property of catalytic SAMs, in which catalysis originates from the cooperative effect between two neighboring catalytic units. The multivalency of the system causes an increase of the number of potential dimeric catalytic sites composed of two catalytic units as a function of the x(1) , which causes an apparent increase in binding affinity (decrease in K(M)). Simultaneously, the k(cat) value is determined by the number of substrate molecules bound at saturation. For values of x(1) >0.4, isolated catalytic units are no longer present and all catalytic units are involved in catalysis at saturation. Importantly, the observed trends are indicative of a random distribution of the thiols in the SAM. As indicated by the theoretical analysis, and confirmed by a control experiment, in case of clustering both k(cat) and K(M) values remain constant over the entire range of x(1) .