Model complexes for phosphodiester hydrolysis

Certain experimental techniques, such as X-ray spectroscopy or mutation analyses of amino acids, which were supposed to play crucial roles in the active sites of nuclease enzymes, contributed to the understanding of the hydrolysis mechanism described above. This paved the way for the synthesis of model complexes, which reduce the enzyme to its essential part required for hydrolysis; the catalytically active core.^[38] These models spread further light on the understanding of the metal and ligand patterns while detailed kinetic studies are easily accessible due to the reduced size of the active center with a manageable number of inorganic scaffolds and ligands.

Numerous artificial metallonuclease model-complexes are found in literature.^[6,39,40] Most of them transpose the parameters that specify naturally occurring enzymes, such as the attendance of a multinuclear metal center with distinct distances between the latter ions in order to bind and activate the substrate and to promote water activation.

Several studies on dinuclear model complexes involving transition metals, such as Zn(II), Fe(II/III) or Cu(II) state, so that their catalytic activities are many times greater compared to their mononuclear analogues.^[41] This feature is based on the fact that two connected metals ions can much better lower the pKa of a water molecule in order to generate the hydrolytically active hydroxide nucleophile under physiological conditions. Moreover, dinuclear metal complexes show an increased activation ability of the bound phosphate esters due to multielectron-transfer processes taking part between the metal sites and the substrate.^[42]

Another vital aspect for the design of these complexes is the utilization of homo- or heterodinuclear metal combinations. Heterodinuclear metal complexes are found for example in purple acid phosphatases (PAPs), a class of enzymes which perform mono-phosphate ester cleavage at low pH values between 5 – 6.^[43] An Fe(III) ion is found in all types of PAPs and is also responsible for the deep purple color caused by a ligand-to-metal charge transfer from the tyrosinate residue to the Fe(III). The occupation of the second metal ion at the active site is dependent on the organism in which the enzyme can be found. In mammalians, a redox active homodinuclear but mixed-valent Fe(III)/Fe(II) complex is found as catalytically active species (Figure 1.10a). In contrast, plant PAPs have an active site composed of either an heterodinuclear Fe(III)/Zn(II) couple or an Fe(III)/Mn(II) couple (Figure 1.10b).^[44]

Model complexes of mammalian PAPs with an active mixed-valent Fe(III)/Fe(II) site are rare due to difficulties in avoiding the formation of the oxidized, and therefore, inactive Fe(III)/Fe(III) couple.^[45] Much better results have been achieved in the synthesis of heteronuclear plant PAP-models by using an unsymmetrical coordination sphere tailored to the individual characteristics of each metal ion in order to regioselectively coordinate the latter.^[46] It had been found that Fe(III) prefers rather “hard” donor ligands, and therefore, resides in an oxygen-rich environment composed of carboxylate side chains of aspartic acid residues and a phenolate side chain of tyrosine. In contrast, Zn(II) or Mn(II) prefer a “softer”

environment with an additional N-donating ligand, such as histidine, as well as with an asparagine residue instead of an anionic aspartate ligand. Some promising PAPs model complexes were successfully synthesized transposing the aforementioned characteristics and being used to evaluate the role of each individual metal in the hydrolysis reaction (Figure 1.11).^[47,48] Furthermore, ligand B is one of the most comprehensively studied mimic of the mixed valent mammalian PAPs. It provides a soft coordination site (N3O3-coordination)

(a) (b)

Figure 1.10 Active sites of mammalian PAPs (a) having a homo-dinuclear Fe(III)/Fe(II) couple (from uteroferrin) and plant PAPs (b) with heterodinuclear Fe(III)/Zn(II) couple (red kidney bean).

suitable for Fe(II) and a hard coordination site (N2O4-coordination) for Fe(III). The metal-metal distance of the acetate bridged complex was determined to be 3.48 Å that is very close to the 3.31 Å reported for natural uteroferrine derived PAPs. However, the mechanism of substrate hydrolysis is still under debate due to opposite opinions regarding the origin of the attacking hydroxide species. On the one hand, the latter species is supposed to be terminally coordinated to the Fe(III) ion, from where it attacks the phosphorus atom. On the other hand, some approaches gave rise to the assumption that it might be bridged between the two metal ions.^[47,48]

Figure 1.11 Examples of model complexes mimicking the active site of PAPs.^[47,48]

However, model complexes for both, heterodinuclear and homodinuclear as well as homodinuclear but mixed valent metals were successfully synthesized. In many cases, the metals are coordinated by a scaffold, which is based on di-ortho-substituted phenols (Figure 1.12, C+D).^[49] The phenolate acts as bridge between the two metal ions, which are held together by two tridentate ligands attached to both ortho positions of the aromatic ring (Figure 1.12). A different scaffold is based on substituted pyrazolates (Figure 1.12, E), which comprise the advantage of an adjustable metal-metal distance by the modulation of the attached tridentate ligands.^[50] These can either be adjusted by different spacer lengths between both moieties or by the generation of an asymmetric coordination sphere.

Figure 1.12 Multidentate ligands for the generation of dinuclear metal complexes. Phenol-based ligands (C and D) with two modular compartments bridged by the phenolate oxygen atom. Pyrazolate-based ligands (E) providing a tunable metal-metal distance depending on the topology of the attached ligands.^[50,51]

The development of biomimetic model systems for naturally occurring metallonucleases over the past decades has made enormous efforts in uncovering the structural and functional patterns of this class of bio-catalysts. The use of different scaffolds as well as the attachment of a variety of ligands contributed to the understanding of the structural compositions of divers active sites of enzymes. Moreover, the individual roles of each metal ion with regard to substrate binding and activation as well as their functions in the hydrolysis mechanism were accessible. However, the models predominantly lack of a comparable catalytic activity, which is still many orders of magnitudes lower with regard to their natural paragons.^[51] The mimic of heterometallic complexes is particularly challenging due to the consideration of metal dependent characteristics, which are important for a site-specific coordination. In addition, the influence of small molecules, such as transient bridging or non-bridging hydroxide nucleophiles or hydrogen-bonding substituents, is still under evaluation. Nonetheless, the reduction of a large peptide to its catalytically active site enables new areas of applications.