The Selection of Amino Acids for the Ligand Design

3.4.5.1 General Strategy

First, the tridentate ligand was retrosyn-thetically separated into his two main com-ponents, see Figure 85. The general ligand design 152 was simplified to two fragments resulting in 2-methylpyridine (153) and the amino acid fragment (154). 153 remains unmodified in the intended ligands and the focus was set onto the amino acids and their residues. Thus, to solve the introduced is-sues, see chapter 3.4.4.2, the relevant ami-no acids were compared regarding their distinct characteristics. Moreover, the pyri-docarbazole ligand and the metal core were defined as structural anchors remaining un-touched.

Figure 85: Fragmentation of the tridentate ligand scaffold.

3.4.5.2 Selection Criteria

The estimated accessible space in the binding site of PI3K has to be assessed to obtain a first hint for the ligand design. Thus, coordinates for the pyridocarbazole ligand and the metal core were extracted from the PI3K (pdb: 3CST) crystal structure and predefined as template structures. All amino acids of the primary sequence of PI3K in 4 Å distance to the pyridocarbazole and the metal core were identified and respected as binding site of the pyridocarbazole moiety.

Two different anchor points were de-fined as A1 and A2. The coordinates of these two anchor points were extrapolated from the crystal structure of -(S)-106, see Figure 59, as an octahedral template in op-posite to 140. These anchor points occupy approximately the same positions as the corresponding coordinating atoms of the tridentate proline-based ligand of -(S)-106.

The distance was set to 2 Å, in congruence to -(S)-106, and the anchor points are in the same plane as the pyridocarbazole lig-and. Thus, the anchor points A1 and A2 represent the positions, where the coordi-nating atoms of the intended ligands should be located. Then, the centre Z1 was de-fined, whereas A1 is located 2 Å away from Z1, which in turn is located 4 Å away from the metal core; all three of them form a line.

The same is true for Z2 and A2 related to the metal core, see Figure 86 a). The exact coordinates can be extrapolated by vector calculations based on the coordinates for the coordinating nitrogen atoms of the pyri-docarbazole and the ruthenium metal core.

The spheres of Z1 and Z2 were defined with 5 Å diameter, see Figure 86 b). These hypo-thetic spheres represent guidance volumes, which should not be exceeded by the in-tended amino acids. Indeed, a sphere of 5 Å seems to be a proper limit avoiding steric hindrances.

a)

b)

Figure 86: a) Overview of the anchor points A1 to A4 and the two centres Z1 and Z2. A1, Z1, the metal core, as well as the nitrogen atom of the pyridine moi-ety of the pyridocarbazole are all in line. The distance Z1-metal core is 4 Å, the distance A1-metal core is 2 Å. The same is true for Z2 in correspondence to A2 and the metal centre. A1, A2, Z1 and Z2 as well as the pyridocarbazole and the ruthenium core are all located in the same plane. A3 and A4 mark the residual ideal positions for coordinating atoms. b) The zones around Z1 and Z2 include the space within 5 Å and represent an hypothetic guidance volume. This volume should not be exceeded by the intended amino acids for the tridentate ligand synthesis. The PI3Kstructure as well as the pyridocarbazole structure are derived from the data set pdb: 3CST.^[188] The pyridocarbazole lig-and is presented as sticks with the carbon atoms in green. Nitrogen atoms are shown in blue, oxygen atoms in red, fluorine in light cyan, and the ruthenium core in purple. PI3Kis presented as cartoon in wheat.

For a further definition of the desired complex structure two additional anchor points A3 and A4, see Figure 86 a), were defined, which are derived from the residual coordinating atoms in -(S)-106. They de-scribe favourable positions to form an octa-hedral complex. Therefore, ideal poses of the fragments 153 and 154 should adress the anchorpoints A1 and A2, occupying the zones around Z1 and Z2, but not A3 and A4.

This is congruent with a presumed fac-coor-dination. Thus, an estimated space of 65.45 ų (represented by the spheres around Z1 and Z2 with a diameter of 5 Å) should be accessible and therefore considered as guidance for the amino acid selection.

However, it is also important which sphere, around Z1 or Z2, is occupied either by the amino acid fragment 154 or the 2-mehylpyridine moiety 153, see Figure 86.

As the two spheres, in a chiral environment, like the binding site of PI3K, are not equal.

The different fragments will experience dif-ferent interactions, when located either in the sphere of Z1 or Z2. For instance, a bulky amino acid residue, like phenylalanine, tyro-sine, or tryptophane, could hypothetically lead to steric hindrances, when occupying the sphere of Z2. In opposite, the offered space occupying the sphere of Z1 could be sufficient for the mentioned bulky amino acids.^[417–419] In contrast, a polar charged amino acid, could experience high attraction in Z2 by forming a salt bridge or could expe-rience high repulsion due to adverse hydro-phobic interactions or charges of the same polarity.^[418–420]

Further, the chirality at the C of the amino acid influences significantly the globular shape of the entire complex, see also Figure 84. The C atom crucially de-fines the three dimensional space, which is occupied by the corresponding functional group of the amino acid fragment. Thus, whereas a complex based on a (S)-confi-gurated amino acid could hypothetically fit into the binding site, the complex based on the corresponding (R)-configurated amino acid could experience steric hindrances and a subsequent repulsion.

Moreover, desolvatation effects of the amino acid residues have to be considered.

Thus, stripping off the hydrate shell of charged or polar groups could significantly decrease the binding affinity towards PI3K, if the polar or ionic group is not captured by a sufficient counterpart inside the binding pocket.^[421] Analogous principles are true for hydrophobic side chains and aromatic side chains. ^[417–419] In contrast, if they find a suf-ficient grove to displace water molecules and to meet hydrophobic or aromatic inter-actions a valuable contribution to the binding affinity could be achieved. However, amino acids with large hydrophobic and aromatic residues result into bulky complexes, which in turn possess decreased water solubility.[140,156,157,422] They require an in-creased amount of solvation mediators like dimethylsulfoxid for in vitro assays. Howev-er, an excessive use of dimethylsulfoxid influences the structural integrity of proteins on secondary, tertiary and quartery level leading to falsified assay results.^[423]

Figure 87: Possible placements of the fragments 153 (a) or 154 (b) into the sphere around Z1. a) 153 occupying the sphere of Z1 as a rigid fragment experiences different interactions in a chiral environment like the binding site of PI3K as occupying the sphere of Z2. b) the same is true for 154. For both fragments the placement into the sphere of Z1 is shown but the examples are analogously true for the placement into sphere of Z2.

Thus, a simple selection on steric crite-ria of the amino acids compared to the cessible space of the binding site is ac-ceptable but not entirely sufficient. In addition, functionalised side chains of amino acids require the application of protection group chemistry to avoid interfering coordi-nation during the complexation reaction.^[416,424] These protection groups have to be planed orthogonal to the residual reaction sequence to ensure a cleavage after a certain planned step.

Therefore, a first generation set of ami-no acids with distinct characteristics to cover the mentioned aspects were selected.

Moreover, both positive and negative con-trols were covered, see also Table 2. These amino acids should ideally act as represent-atives for related ones, i.e.: phenylalanine as representative for aromatic amino acids.

In addition, to minimise protection group chemistry, amino acids with ideally ortho-gonal protectable functional groups regard-ing the complex synthesis were preferred.

Thus, the first representative amino acid group consisted of L-alanine ((S)-155),

D-alanine ((R)-155), and L-serine ((S)-159) as small sized ones. These amino acids, regarding their VAN-DER-WAALS volume, should hypothetically fit into the binding site.

This is only true if the estimated accessible volume of approximately 65.45 ų of Z2 complies to the existing conditions of the PI3K binding site. Moreover, the influence of the C stereoconfiguration, during these investigations, should be covered comparing both L-alanine ((S)-155) and D-alanine ((R)-155).

The second group was represented by

L-phenylalanine ((S)-156), L-leucine ((S)-158), and L-valine ((S)-160). These large unpolar amino acids should result into bulky complexes experiencing steric hin-drances. Thus, they should subsequently possess a reduced affinity towards the PI3K binding site. Due to the rotation around the C and the C bond, the bulky

residues of the members of this group could potentially avoid steric clashes. To investi-gate the elimination of this rotational free-dom D-phenylglycine ((R)-179) as a non-coded amino acid was added to this group.

As a third group L-histidine ((S)-157) and L-tyrosine ((S)-161) were selected rep-resenting large aromatic but simultaneously polar amino acids. As for the second selec-tion group, the resulting complexes should hypothetically be excluded from the binding site by steric hindrance. Thus, the resulting complexes should also represent negative controls.

As the last group, L-proline ((S)-101) as well as D-proline ((R)-101) were applied again as established ligand systems due to their successful former application, see Chapter 3.3.2.

Table 2: Amino Acid Characteristics

Amino Acid Scaffold Charge Polarity 3 vdW Volume [Å] Hydrophobic Index

Alanine aliphatic neutral apolar 67 1.8 Arginine aliphatic basic polar 148 -4.5 Asparagine aliphatic neutral polar 96 -3.5 Aspartate aliphatic acidic polar 91 -3.5 Cysteine aliphatic neutral polar 86 2.5 Glutamate aliphatic acidic polar 109 -3.5 Glutamine aliphatic neutral polar 114 -3.5 Glycine aliphatic neutral apolar 48 -0.4 Histidine aromatic basic polar 118 -3.2 Isoleucine aliphatic neutral apolar 124 4.5 Leucine aliphatic neutral apolar 124 3.8 Lysine aliphatic basic polar 135 -3.9 Methionine aliphatic neutral apolar 124 1.9 Phenylalanine aromatic neutral apolar 135 2.8 Proline heterocyclic neutral apolar 90 -1.6 Serine aliphatic neutral polar 73 -0.8 Threonine aliphatic neutral polar 93 -0.7 Tryptophan aromatic neutral apolar 163 -0.9 Tyrosine aromatic neutral polar 141 -1.3 Valine aliphatic neutral apolar 105 4.2