MESA+ Institute for Nanotechnology

Supramolecular catalysis is the main topic of this thesis. New calix[4]arene-based metallo-catalysts were synthesized by linking one, two or three ligating groups at the upper rim of the calixarene skeleton. The catalytic activity of the copper, zinc or barium complexes was investigated in phosphoryl and acyl transfer reactions.

Previous research had revealed that the calix[4]arene motif is an interesting scaffold to build multifunctional enzyme models, thanks to its ability to opportunely preorganize the catalytic functions at the upper or lower rim. Moreover, even when blocked in the cone structure, it maintains a residual conformational mobility, and this allows the calixarene based catalysts to adapt themselves to the changes of the substrate geometry in the transition state. An important aspect is the development of water-soluble catalysts. For this purpose a supramolecular approach was used, which exploits the interaction between adamantyl calix[4]arenes and β-cyclodextrins (β-CD). The interaction between some of these calixarene derivatives and polyvalent β-CDs and β-CD self-assembled monolayers (SAMs) was also studied in solution and at surfaces, respectively.

A general introduction to enzyme catalysis and its origins is reported in the first part of Chapter 2. The mechanisms of the phosphoryl and acyl transfer reactions, with particular attention to the role of metal ions, are explained in the second part of this chapter and examples of natural nucleases and peptidases that exploit divalent ions are described. Recent examples of synthetic mono-, di- and trinuclear metal catalysts for the cleavage of phosphates, amides and esters are reviewed in the third part, underlining the factors that influence the cooperativity of the metal ion centers.

Chapter 3 describes three different approaches to solubilize in water a calixarene compound bearing three 2,6-bis[(dimethylamino)methyl]pyridine groups at the upper rim. Its zinc complex had previously shown good catalytic activity in the phosphodiester cleavage in EtOH/H2O 35%. Water solubility is an essential property of nuclease mimics because, ultimately, their activity has to be studied on natural substrates such as RNA under physiological conditions. In order to make this compound soluble in water we introduced adamantyl units at the calixarene lower rim and exploited the formation of inclusion complexes with β‑CDs in water. It was found that the adamantyl group (Ad) is incompatible with the reactions needed to attach the pyridine units at the upper rim. However, it was possible to prepare some water soluble p‑guanidinium di- and tetraadamantyl calix[4]arenes and to study the calix[Ad]‑ β-CD interactions in solution by microcalorimetry and at the surface of β-CD SAMs by surface plasmon resonance. When the spacer between the Ad group and the calixarene is more than twelve atoms, all four adamantyl moieties can be included by β‑CDs without interference among binding sites, and with intrinsic binding constants in the range 2.9‑5.4x104 M-1. Another approach towards a water-soluble calixarene-based catalyst consisted in the introduction of six hydroxyl groups at the upper rim of a trispyridine catalyst, obtaining a compound with a solubility of 2x10-4 M. However, studies of Zn(II) complexation in pure water revealed that the binding constant of the 2,6-bis[(dimethylamino)methyl]pyridine chelating units is too low to ensure a quantitative formation of the complex at millimolar concentration.

In Chapter 4 the procedure to obtain a calix[4]arene with two functional groups such as alcohols, formyls or carboxylic acids in the 1,2-proximal positions is described. These compounds are important intermediates for the synthesis of catalysts bearing metal ions in proximal positions. Novel difunctional calix[4]arene ligands were prepared by introducing two monoaza-18-crown-6 in the 1,2-proximal or 1,3-diametral positions of the calix[4]arene upper rim. The Ba2+ complexes of these compounds were investigated as catalysts in the ethanolysis of esters endowed with a carboxylate anchoring group. For the first time a direct comparison between diametral and proximal calix[4]arene supramolecular catalysts was performed. Even if in both cases the metal centers are able to cooperate, the proximal calixarene catalyst is largely superior to its diametral regioisomer. One of the parameters that mostly influence the reactivity is the distance between the carboxylate and the ester carbonyl groups, but other factors, favoring the proximal catalyst, are also involved.

Chapter 5 describes, in the first part, the synthesis of a calix[4]arene equipped with two 2,6-bis[(dimethylamino)methyl]pyridine ligating groups in the proximal positions in order to compare the catalytic activity with those of similar catalysts reported in the literature. In the second part, the synthesis of a new class of ligands ([12]aneN3-calixarenes) is described. These compounds were synthesized by functionalizing the calix[4]arene upper rim with one, two (in diametral or proximal positions) or three triazacyclododecane ([12]aneN3) macrocycles. The [12]aneN3 is a ligating group for divalent transition metal ions which is much stronger and more hydrophilic than the 2,6-bis[(dimethylamino)methyl]pyridine, thus allowing a higher stability and an easier solubilization in water of the calixarene metal complexes. The catalytic activity of the Zn(II) complexes of the pyridine-calixarenes and [12]aneN3-calixarenes was investigated in the methanolysis of aryl esters. The measurements, carried out in methanol at pH 10.4, show that the two classes of catalysts have similar catalytic properties. The presence of a carboxylic anchoring group on the substrate is necessary to obtain high rate enhancements, which, in some cases, reach values up to 105 in the presence of 1 mM catalyst. The 1,2-proximal calix[4]arene complexes are much more effective than their 1,3-diametral isomers. The two metal centers in proximal positions can cooperate better in the catalytic process, as one coordinates the carboxylate group present on the substrate and the other activates the attacking methoxide anion. The higher rate enhancements obtained with the trinuclear catalysts were explained by taking into account the statistical advantage of these compounds over the corresponding 1,2-dinuclear ones. Only in the case of the trinuclear Zn(II) pyridine-calixarene complex with the p-carboxyphenyl acetate a significant contribution of the third Zn(II) ion in the activation of the ester carbonyl group was found. The Zn(II) complexes of the 1,2-proximal disubstituted and trisubstituted [12]aneN3-calixarenes, show a remarkable shape selectivity for m-carboxyphenyl acetate over its para-isomer. The reactions follow Michaelis-Menten kinetics with the formation of a catalyst-substrate complex that reacts to give the product.

Chapter 6 reports, in the first part, the activity of Zn(II) and Cu(II) [12]aneN3-calixarene complexes in the cleavage of phosphodiester bonds in the RNA hydroxypropyl-p‑nitrophenyl phosphate ester (HPNP). The choice of copper(II) metallo-catalysts was due to the fact that [12]aneN3-calixarene Cu(II) complexes are more soluble in water and show higher cooperativity in the activation of the phosphate bond of HPNP compared to the corresponding Zn(II) complexes. The rate enhancement of HPNP transesterification catalyzed by the trinuclear Cu(II) calixarene measured as a function of the substrate concentration showed Michaelis-Menten kinetics, where the substrate binds to the catalyst with a Kass = 500 M-1 and it is converted into product with a kcat = 7.6 x 10-4 s-1. In the second part of the chapter, the catalytic activity of the Cu(II) trinuclear complex in dinucleotide cleavage is reported. A remarkable selectivity for UpU and UpG dinucleotides was observed, e.g. UpU is cleaved 40 times faster than GpA. Compared to the uncatalyzed reactions, the rate enhancements measured for the cleavage of UpU and UpA are in the order of 7 x 103.

In Chapter 7 the catalytic activity of Cu(II) [12]aneN3-calix[4]arene complexes in oligonucleotide cleavage was investigated. The reactions were carried out at 50ºC, [cat] = 50 μM, and the substrates were six-, seven- and seventeen-base oligomers radiolabeled with 32P in 5’-position. Both the two dinuclear and the trinuclear copper complexes are very efficient catalysts in the cleavage of phosphodiester bonds within an oligomer. Beside the cooperative action of the metal centers, also the calixarene apolar skeleton seems to play an important role in the catalytic process. Its action is probably due to the creation of an apolar environment around the oligonucleotide that favors hydrophobic interactions along with hydrogen bonding useful for the phosphate cleavage. Phosphodiester bonds having a pyrimidine nucleotide in the 5’-position are cleaved faster. In all the investigated cases, CpA is the most reactive bond with all the catalysts, and the ease with which this bond can be cleaved depends also on its position in the oligonucleotide, increasing when the bond is closer to the 5’-terminal position. The results indicate that the reactivity and selectivity data obtained with RNA models or dinucleotides cannot be simply extended to larger oligonucleotides or RNAs, since with macromolecular substrates many other factors could come into play.