The catalytic efficiency, mechanistic pathways, and structural complexity displayed by enzymes make them a tremendous source of inspiration for chemists involved in catalyst development[1, 2]. Nature has evolved enzymes as large multi-kilodalton complex structures in which even units that are remote from the actual active site may profoundly affect the activity of the enzyme[3]. The much lower complexity of artificial enzyme mimics may be an important reason for their typical modest performances with respect to enzymes. This awareness has led to an interest in catalysts based on multivalent scaffolds, such as dendrimers[4], micelles[5], and nanoparticles[6], with the idea of increasing the structural complexity of the synthetic system. A key challenge is straightforward access to synthetic catalysts that can match up to the size and complexity of enzymes. The necessity for multistep synthesis can be overcome by relying on self-assembly for the formation of the multivalent structure. In particular, the self-assembly of catalytic monolayers on the surface of gold nanoparticles (AuNPs) to give gold monolayer-protected clusters (AuMPCs) is emerging as an attractive strategy[7, 8]. Nonetheless, although they rely on self-assembly, the composition of self-assembled monolayers (SAMs) on Au NPs is typically still of rather low complexity[9]. Varying the surface composition by thiol clustering is a very difficult approach because this does not give a full control over the final composition, requires purification of each single NP system, and suffers from issues related to the characterization of mixed SAMs both in terms of composition and morphology. As a consequence, enormous efforts should be done to purify the different species coming from the synthesis. For that reason, in the recent years a new approach emerged by using nanoparticles not as direct tools, but as scaffolds to bind secondary molecules on.
Rotello and coworkers were the first to realize this pioneering idea showing the attractiveness of cationic Au MPCs as a construction element for the development of innovative biosensors[10]. Inspired by those contributions, Prins et al. recently started to study the formation of heterofunctionalized multivalent structures relying on the self-assembly of small anionic peptides on the surface of Au MPCs[11, 12]. The results showed that it is possible to control the peptide surface composition simply exploiting the different affinities for the cationic monolayer. As a consequence, the low complexity of the surface can be overcome.
This concept has been further developed during the PhD-project[13]. Based on the self-assembly of oligo-anions on a cationic surface, we went beyond the simple surface composition control and developed a true supramolecular nanoenzymatic system. It is composed by two fundamental elements:
1. Gold nanoparticles functionalized by alkyl thiols featuring a Trimethylammonium group in the Ω-position (8-Trimethylammonium octylthiol NR4+-AuNPs) thus generating a cationic surface;
2. Anionic oligopeptides that bind the nanoparticles’ surface. These feature a C-terminal tail with three Aspartic acid residues; an N-terminal tail composed by a variable number of Histidines (H0-H3); a central Tryptophane residue linking the two edges (Ac-HnWDDD-OH). The four negative charges coming from the C-terminal tail give an efficient binding, while Histidine and Tryptophane allow, respectively, catalysis and signal output for peptide concentration and binding measurements.
Both of these constitutional elements are inefficient catalysts by themselves. Only when the peptides self-assemble on the cationic surface an active system is formed, which is able to accelerate the hydrolysis of N-CBz-(D)Phe-ONP by two orders of magnitude over the background. Importantly, the multivalent surface plays a crucial role in tuning the catalytic activity. The surface not only brings the substrate and catalyst in close proximity but also generates a microenvironment with an enhanced local pH that further activates the catalytic peptide. Given the supramolecular nature of the whole system and considering what has been written about the fine regulation of the surface composition, this system is highly adjustable simply by modulating the concentration of the constitutional elements that self-assemble on the surface.
Once we obtained the lead system we started to investigate the intrinsic features that characterized it like the importance of the chemical structure of the substrate, the peptide catalyst and the cationic surface.
The sequence of the peptide catalyst is very important for the efficiency of the catalytic system. Mutations on the H1 sequence (Ac-HWDDD-OH) altering its order and length showed that Triptophane has a subsidiary role in binding and its position should be next to the C-terminal tail. Histidine should occupy the N-terminal position because the presence of other residues in such position would reduce the catalytic efficiency of the Imidazole residue. If the sequence is extended by insertion of a Glycine residue between the Tryptophane and the C-terminal tail, we still observe a lower hydolysis rate, but not so drastic as the one observed by flipping the N-terminal sequence (steric hinderance).
Like a natural enzyme, this system has specific requirements for the substrate that undergoes hydrolysis. SAR studies have been performed on substrate analogs with different Nα-protecting groups and side chains. The results showed that large and hydrophobic substrates have a higher affinity for the nanoparticles and are hydrolyzed faster. This is presumably due to interactions between those hydrophobic surfaces and the hydrophobic part of the monolayer. The aromatic substrates are favoured respect to the alkyl ones which emerges from the comparison between the kobs of N-CBz-Leu-ONP and N-CBz-(L)Trp-ONP: the latter is hydrolyzed much faster. The Nα-protecting group seems to have a crucial role in the substrate stabilization: diminishing its dimensions, thus diminishing hydrophobicity, results in a fast, spontaneous hydrolysis in simple buffer.
A significant effort was made to improve the catalytic performances of the system through a supramolecular approach. In particular a hybrid system was prepared by self-assembling peptide H2 (Ac-HHWDDD-OH) and a library of non-catalytic peptides contemporarily on the monolayer surface (Ac-XXWDDD-OH, where XX are Leu, Phe, Ser and Arg in 42 possible combinations). The idea was that the second peptides would modulate the catalytic efficiency of the system. The results did not match the expectations which is presumably due to the fact that those peptides did not compensate the loss of the pH effect with additional interactions.
Mutations on the H1 sequence (Ac-HWDDD-OH) by inserting identical flanking residues beside Histidine (Ac-XHXWDDD-OH) were studied with the scope of generating enantioselectivity. Although the variety of the flanking residues was large, (Leu, Phe, Ser, Tyr) no substrate enatioselectivity was observed. Despite this we had some experimental evidences that suggested that some enantioselectivity could be obtained by exploiting the binding of the substrate on the nanoparticles surface
