Calix[n]arenes: features and uses in biology

71

72

Figure 3.1.2.2. Synthesis of even-numbered calix[n]arenes.

In addition, calixarenes differentiate from other macrocycles for their versatile conformational properties. In fact, calix[n]arenes (n = 4,5,6,8) with methyl or ethyl groups at lower rim are conformationally mobile and in solution present a mixture of conformers, which can interconvert each other, whereas calix[4]arenes with alkyl chains longer than the ethyl group at lower rim can be blocked in 4 different conformations: 1,3-alternate, partial cone, cone and 1,2-alternate (Figure 3.1.2.3b)^18,19. In the optic of multivalent ligands the possibility to have scaffolds able to orient active units towards different directions is an interesting way to study how simultaneous binding affects recognition of biological counterpart. In doing this, sometimes a rigid and preorganized structure increases the binding efficiency due to the less loss of degrees of freedom, whereas sometimes, instead, mobile conformation can help in adapting the ligand itself to the receptor geometry.

NaOH Ph2O, Δ

p-tert-butylcalix[4]arene η = 61%

p-tert-butylcalix[6]arene η = 83-88%

p-tert-butylcalix[8]arene η = 62-65%

KOH, xylene, Δ

NaOH, xylene, Δ

73

Figure 3.1.2.3. Schematic representation of the a) valencies, b) geometries and c) functionalizations of calix[n]arenes.

Eventually, calixarenes are characterized by two different rims: the lower rim, identified by hydroxyl groups, and the upper rim, determined by the para positions of the phenolic rings. A large variety of groups can be added to these two rims directly or via linker in a selective way (Figure 3.1.2.3c). The length, the nature and the mobility of the spacer must be opportunely modulated, since either take active units at proper distance or participate to the binding. Due to their numerous properties, calix[n]arenes allow to synthesize a library with different conformations, size and type of functionalization exploring a wide chemical space, which could help us more easily find the perfect ligand-protein match.

Figure 3.1.2.4. A) guanidine calixarenes for Shaker potassium channels stoppering and p53 stabilization; B) glycocalix[5]arene for multivalent inhibition of Cholera toxin.

This versatility could be exploited towards proteins to increase efficiency and specificity of binding phenomena allowing us to modulate or even stop pathological pathways. Several are the examples present in literature about the employment of functionalized calixarenes as multivalent ligands. A library of tetraguanidinium cone calix[4]arenes (Figure 3.1.2.4A) was synthesized to inhibit voltage-dependent potassium channels²⁰, mainly Shaker potassium channels, which are involved in different pathologies. It has been demonstrated that some

Calix[4]arene Calix[5]arene Calix[6]arene Calix[8]arene

Upper rim functionalized Lower rim functionalized Cone Partial cone 1,2-alternate 1,3-alternate a)

c) b)

A B

74

compounds can reach even an inhibition constant of 50M, whereas the monomeric analogue is not active. The mechanism of action is still under investigation, but it is clear that it is supported by a multivalent effect. Similar compounds were used also in the stabilization of p53 tetramer required for activation of repair machinery and apoptosis process²¹. In fact, R337H mutation substitutes a permanently positive charged arginine with a histidine, which at physiological pH is half protonated and so physiological interactions with E336 and E339 are obviously weakened. Some of the compounds showed in Figure 3.1.2.4A have been shown selective to the mutated form and efficient binders at 400 M. Supporting molecular studies demonstrated that guanidine calixarenes can bind to both sides of the protein complex via multiple Arg-Glu interactions, which implies that multivalency is exploited²². The best multivalent effect has been obtained with glycocalixarenes for the Cholera toxin (CT) inhibition²³. CT is characterized by AB5 structure, in which B units present epitopes selective to pentasaccharide GM10, localized on cell surface. In order to stop the relative infection, in Sansone’s laboratories was developed calix[5]arene derivative decorated at the upper rim with 5 units of GM10 in order to mimic the B5 substructure of CT (Figure 3.1.2.4B). Through ELISA tests was determined an IC50=450pM and in comparison to the monovalent molecule, an amazing multivalent effect of 20000.

3.1.3 Multivalency in CA inhibition

As already largely discussed in Chapter 2, a wide plethora of small molecules with different scaffolds, tails and heads have been proposed and studied for CA inhibition. They are designed to make contacts inside the catalytic active site of the enzyme forming a 1:1 complex. However, as more widely anticipated previously, in recent years some research groups started to explore the possibility of exploiting multivalent ligands also for the inhibition of enzymes although these are mostly characterized by the presence of a single active site. The idea is to obtain an enhancement of the inhibition activity on the basis of a binding-rebinding process favoured by an increased local concentration of the ligand. In this context one of the most important class of enzymes tested for the inhibition by multimeric molecules is that of glycosidases. Towards them, some particular scaffolds showed to be able to exploit multivalency (even reaching rp/n=4800), in which the multiple exposition of the same active unit on a preorganized scaffold significantly increases dramatically their affinity for the biological counterpart with respect to the monovalent analogue.

Moreover, in Nature some CA isoforms are present in a multimeric assembly (e.g. dimeric VchCAβ). Therefore, towards these particular cases, multivalent ligands could put into play a real multivalent effect as that possible with oligomeric carbohydrate recognition proteins (e.g.

lectins). If many examples are indeed known of high-efficiency multivalent ligands for non-enzymatic proteins, only a few preliminary examples are reported towards non-enzymatic proteins.

In the attempts of multivalent inhibition of CAs, some preliminary studies have been carried out exploiting well-known multimeric scaffolds. Up to now, in this avant-garde idea PAMAM dendrimers and fullerenes were tested.

In Carta’s work²⁴, has been shown how PAMAM derivatives can be very versatile scaffolds for the development of multivalent inhibition of CAs. Three generations of PAMAM derivatives were synthesized, which expose up to 32 benzenesolfonamide units per PAMAM core. Besides

75

their exceptional inhibition properties towards hCAI, hCAII, hCAIX and hCAXII, they have been shown as potent multivalent inhibitors. In particular, PAMAM G3 (Figure 3.1.3.1A) towards hCAII showed a relative potency normalized to the number of sulphonamide units (rp/n) of 285.7 nM, which shows a significant multivalent effect. Some of these derivatives, moreover, showed interesting properties in lowering intraocular pressure in glaucomatous animal models by inhibiting the hCAII and hCAXII enzymes present within the eye.

Figure 3.1.3.1. A) PAMAM G3 derivative functionalized with benzenesolfonamides, figure adapted from reference²⁴, B) fullerene derivatives functionalized with coumarins, figure adapted from reference²⁵. Since fullerene-based multivalent molecules were shown in the past to be very efficient multivalent inhibitors towards α-mannosidases, Abellan-Flos and al.²⁵ tried to use multivalent coumarin-functionalized fullerenes as potential multivalent inhibitors towards CAs. They synthesized and studied different C60 fullerenes, in which twelve coumarin units were grafted on them by different spacers (Figure 3.1.3.1B). What turned out is that these structures are able to inhibit very efficiently CAs (in particular the extracellular ones), but without exploiting a clear significant multivalent effect.

Supuran and co-workers²⁶, as well, tried to decorate AuNPs with benzenesolfonamide moieties, but also in this case, despite their high efficiencies, they did not show a significant multivalent effect. Probably the high bulk of these systems and the crucial nature of spacers are responsible for a non-exploitable multivalent effect.