Cleavage of phosphate diesters by functionalized gold nanoparticles: from simple models to plasmid

For many decades, scientists have attempted to understand and replicate nuclease efficiency. These enzymes are responsible for the cleavage of phosphodiester bonds, bonds that constitute the backbone of nucleic acids. By studying nuclease active sites, as well as reported examples of artificial enzymes able to cleave DNA and RNA models, chemists aim to achieve systems possessing better catalytic properties. Designing artificial nucleases which can overcome the structural complexity and compete with the efficiency observed in Nature’s enzymes can be achievable with complex, nevertheless easy to prepare architectures, like gold nanoparticles (AuNPs). Indeed, functionalized AuNPs are one of the most attractive architectures reported so far in terms of phosphodiester cleavage. The necessity for a multistep synthesis can be easily overcome by using thiols as passivating agents that strongly interact with the metal cluster. By precisely tuning the organic thiol structures, it is possible to modulate AuNPs functionalization and properties of the reaction environment. Moreover, taking advantage of supramolecular chemistry principles, multivalency of gold nanoparticles may provide the cooperativity of several active and identical groups, enhancing the catalytic efficiency. Herein, studies on the cleavage of DNA and RNA models, such as: 1) 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) 2) bis-p-nitrophenyl phosphate (BNP) 3) uridine 3’-(LG) phosphates and easy to manipulate DNA (plasmid pBR322 DNA), using self-assembled gold nanoparticles (AuNPs) are presented. Five different catalytic systems (AuNP1-5) were functionalized with at least one zinc complex of 1,4,7-triazacyclononane (TACN), either connected to a hydrocarbon chain (AuNP1,2) or decorated with different flanking species (a second Zn(II) complex, triethyleneoxymethyl derivative or the guanidinium unit of arginine; AuNP3-5). In particular, chapters 1-3 describe the fundamental aspects of phosphates and nucleic acids, as well as cover the most relevant features contributing to the acceleration of the hydrolysis of nucleic acids by artificial nucleases. Furthermore, the principles of gold nanoparticles, including their properties, synthetic methods, application, as well as techniques of their characterisation are described. Chapter 4 covers the synthesis and kinetic studies on the hydrolysis of 2-hydroxypropyl p-nitrophenyl phosphate. Performed Michaelis-Menten-like kinetics, Zn(II) titration, pH-profiles, kinetic isotopic effect as well as competitive inhibition with dimethyl phosphate enabled the proposal of two mechanisms towards the cleavage of HPNP, based on type of catalytic units present on the AuNP monolayer. Studied AuNPs were able to hydrolyse HPNP almost 1 million times compared to background reaction, being so far one of the most efficient systems cleaving this RNA model substrate. Chapter 5 reports the results of the cleavage of bis-p-nitrophenyl phosphate and pBR322 DNA, as a DNA model substrate (the former one) and an example of a real polymer (the latter one). Thorough studies revealed significant differences in AuNPs catalytic activity in the hydrolysis of BNP and plasmid DNA. Among all studied nanozymes, only a Y-shaped species decorated with a peptide comprising serine and arginine in its sequence (AuNP4) was able to efficiently convert the supercoiled DNA into its nicked form in less than 3-4 hours at 35 μM concentration. Interestingly, in the hydrolysis of much simpler model BNP, the most reactive were nanozymes AuNP1-3, displaying only a minimal efficiency in the plasmid DNA cleavage. Chapter 6 reports studies dedicated to the leaving group departure from uridine 3’-(LG) phosphates with the most efficient in HPNP and BNP cleavage, AuNP1. Final results of the hydrolysis of chosen nucleotides were presented in the form of Brønsted plot, displaying no change throughout the explored pKa range of leaving groups. Furthermore, a great negative value of the Brønsted constant suggested a significant development of anionic charge in the transition state (βLG = -0.86). Taking advantage of the reactivity and ease in monitoring the cleavage of uridine 3’-p-nitrophenyl phosphate, more detailed UV-Vis-based kinetic studies in the presence of either AuNP1 or the zinc complex of 1,3-di(1,4,7-triazonan-1-yl)propan-2-ol Zn2(4e) were performed, providing additional information about Zn(II)-TACN interactions with uracil. Moreover, the reactivity and catalytic properties of AuNP1 vs Zn2(4e) in nucleotide cleavage revealed that, due to conformational flexibility, nanoparticles 1 were able to easily accommodate bulky substrates, contrary to what happens with the rigid Zn2(4e). The presented supramolecular systems appear to be among the best reported catalysts for the cleavage of studied nucleic acids model substrates. Mechanistic investigations afforded a detailed information of the way AuNPs hydrolyse phosphodiester linkages. It has been demonstrated that the proper design and synthesis of nanoparticles may embrace significant factors that make nucleases one of the most efficient enzymes encountered in Nature.