Ascorbate Reduction as Extent of the Ligand Interception in ROS Origin

To study the capacity of the ligands to intercept in a Cu^2+/+ redox cycle, analogous to the one which is proposed for ROS generation in AD^[173], time dependent ascorbate consumption was monitored by UV spectroscopy. The standard set up for the experiment was 100 μM sodium ascorbate, 5 μM Cu²⁺ and 7 μM of ligand or Aβ1-16 in 50 mM phosphate buffer. The order of addition was buffer, ascorbate, ligand/protein, copper. The ascorbate stock solution was freshly prepared and no longer used than 5 h. The excess of ligand was used in order to make sure that no free Cu⁺ is present. Ascorbate consumption was followed over a time period of 5 minutes and evaluation was graphically done by plotting the absorption (265 nm, ԑ = 14500 M^-1 cm^-1) against time. The metal becomes re-oxidised by O2 from the air. The experiment was performed with both the methylated and the not methylated ligands to verify the results obtained in the Fz experiment (chapter 6.2). Each measurement was repeated at least three times to minimise errors and quantify the results. The detected ascorbate consumption should be slow in case of a strong Cu+ chelator and fast in case of weakly coordinating ligand systems. Thus, the following order could be assumed according to the determined stability constants: L^6H (slow) < L^8H < Aβ1-16

<< L^3H ≈ L^5H (fast).

Figure 37 shows the time depended ascorbate consumption for the methylated and not methylated ligand systems. Between the methylated and the not methylated systems no significant difference could be recorded. The biggest deviation could be observed for L^3H and

Aβ1-16

Aβ1-16 + Cu⁺ Aβ1-16 + Cu⁺ + L^8H

72 determined stability constants only a small inhibition of the metal mediated redox cycle.

-1,2

Figure 34 Time depended ascorbate consumption at 265 nm. Conditions were 100 μM sodium ascorbate, 5 μM Cu²⁺

and 7 μM L^X or Aβ1-16 in50 mM phosphate buffer (pH 7.4). Left the not modified ligands and right the methylated ligands.

The different inhibition capacities of L^6H/Me compared to the stability constants can be explained by different redox pairs formed during the experiment and should also be considered by the interpretation for the other ligand systems(Scheme 20). Aβ1-16 has arelatively strong affinity to Cu²⁺ and binds it in the nM range.^[113,114] Thus, after addition of Cu²⁺,the Cu²⁺(Aβ1-16) complex is formed, which gets reduced by the ascorbate leading to Cu⁺(Aβ1-16). Thus, in case of Aβ1-16 the observed redox pair is [Aβ1-16Cu]²⁺/[Aβ1-16Cu]⁺. The oxidation as well as the reduction requires reorganisation of the protein. The single redox states are therefore stabilised and the re-oxidation hindered. As a consequence the protein shows a remarkably strong inhibition in the ascorbate oxidation. The ligands L^3Me, L^5Me and L^8Me are in contrast only selective for Cu⁺. The observed redox partners are therefore the [L^XCu]⁺ complex and the ligand free [Cu(H2O)5]²⁺

complex and thus the ascorbate experiment resembles the determined stability constants. As mentioned before L^6H can not only coordinate to Cu⁺, but also form a stable complex with Cu²⁺. The formed redox pair is likely 2 [L^6HCu]⁺/[(L^6H)2Cu]²⁺ + [Cu(H2O)5]²⁺. The low activity of L^6H is most likely induced due to the stabilisation of the oxidised [(L^6H)2Cu]²⁺ species. As a consequence the Ligand L^6H shows a higher activity than L^3Me and L^5Me but still much less active than the Aβ1-16, although the determined stability constant is higher.

-1,2

6.4. Ascorbate Reduction as Extent of the Ligand Interception in ROS Origin

73

Scheme 20 Reaction pathways in the ascorbate consumption experiment. L^X can be L^3H, L^5H, L^8H or their corresponding methylated derivatives.

The experiment was repeated at different conditions to further investigate the inhibition capacity of the most active chelator, L^8H. Figure 35 shows the ascorbate consumption at 2.5 μM

and 7 μM in the presence of Aβ1-16 and without. By lowering the concentration of ligand to equimolar concentration of the copper a remarkably increase of the ascorbate consumption could be observed. One reason for this increase could be the presence of free Cu⁺. Even small inaccuracies in the pipettes or minimal impurities in the used compounds could lead to an excess of copper and thus a significant increase in the ascorbate consumption. Further decrease of the ligand concentration, to 2.5 μM,results in a loss of the protective function. Besides low ligand concentration higher concentration were tested. However, increase to 10 μM or higher (15 μM, 20 μM) have no significantly influence on the ascorbate oxidation.

-1,2 without. Spectra were recorded in 50 mM phosphate buffer (pH 7.4).

The experiment was also carried out in the presence of Aβ to study the influence of the protein on the sensitive redox system. The addition of Aβ1-16 leadsto a further decrease of the ascorbate oxidation. One possible explanation for this increased inhibition would be that a ternary complex is formed where the Cu⁺ is tightly boundby the protein and the chelator. However, formation of such a complex is unlikely because it was not observed in the NMR metal exchange experiment.

In Scheme 21 is a proposed redox cycle presented, which comprises all important intermediates and could explain the observed reactivity’s. The redox cycle starts with the free Cu²⁺, which becomes coordinated by the Aβ1-16. In the next step takes the reduction place and generates the [Aβ1-16Cu]⁺ complex. The higher affinity of L^8H should than lead to a replacement of the protein

74

and coordination of the ligand, like observed in the NMR experiment. This [L⁸Cu]⁺ complex becomes then oxidised by air and releases the Cu²⁺, which can again be coordinated by the Aβ1-16. Since no studies were done concerning the kinetics, it can only be conjectured what the rate determining step is. The reduction and oxidation of the metal is more or less known from the previous experiments and in both cases faster ascorbate consumption was observed. The metal exchange rate instead, is not specified. However, it could be seen in the ferrozine experiment (see chapter 6.2) that such a metal exchange can be relatively slow. Thus, the increased inhibition activity is not induced by a stronger coordination, but most likely due to an intermediate were the Aβ1-16 gets replaced by the ligand.

Scheme 21 Proposed redox cycle for the ligand inhibited ascorbate consumption in the presence of Aβ1-16.

The ¹H-NMR experiment showed that L^3Me and L^5Me can, together with Aβ1-16, probably form ternary Cu⁺ complexes. Thus, the ascorbate consumption experiment in the presence of Aβ1-16

was also repeated for L^3Me and L^5Me to studythe influence of these complexes. A higher inhibition capacity was expected, since an additional ligand is involved in the coordination of the Cu⁺. However, the comparison with the Aβ1-16 alone showed no significant deceleration of the ascorbate consumption. This leads to the assumptions that either no ternary complex is formed or that the coordination of the ligand is too weak and does not influence the re-oxidation of the Cu⁺. The latter hypothesis is disfavoured by the fact that the tripodal ligands have already shown an activity. Thus, coordination should induce a change in the inhibition.